Targeted adenovirus vectors for delivery of heterologous genes

ABSTRACT

Modification of internal sites of the adenovirus fiber protein and hexon protein permit effective targeting of adenovirus vectors. Accessible sites to redirect adenovirus targeting were identified. The HVR5 loop of the hexon protein and the HI loop of the fiber protein (knob) were highly permissive for the insertion of foreign protein sequences, which apparently did not impact on the viability and productivity of corresponding viruses. Accessibility and functionality of the epitope strongly depend on the size of the neighboring spacers. Other results suggest that short targeting peptides can be effectively fused to the C-terminus of the fiber protein. In a specific embodiment, a series of adenovirus vectors modified at the HVR5 site, the fiber protein HI loop, or the fiber protein C-terminus to target urokinase-type plasminogen activator receptor bearing cells were prepared. Such vectors are particularly useful for targeting the vasculature, e.g., for gene therapy of cancers or cardiovascular conditions.

FIELD OF THE INVENTION

The present invention relates to effective targeting of adenovirusvectors by modifying surface accessible sites of the fiber or hexonproteins to include a targeting amino acid sequence. The key to successof the present invention lies in the discovery that additional spaceramino acid residues at the N-terminus and C-terminus of the insertedtargeting sequence are critical to providing a recognizable bindingstructure of the targeting sequence. Thus, accessibility andfunctionality of the targeting sequence as well as structure of themodified protein strongly depend on the size and nature of theneighboring spacers. Other results suggest that short targeting peptidescan be effectively fused to the C-terminus of the fiber protein. Theinvention further relates to the use of such vectors for deliveringtherapeutic genes to specific target cells in vitro and in vivo.

BACKGROUND OF THE INVENTION Adenovirus Vectors

Adenoviruses exhibit certain properties which are particularlyadvantageous for use as vector for the transfer of genes in genetherapy. In particular, they have a fairly broad host spectrum, arecapable of infecting quiescent cells, do not integrate into the genomeof the infected cell, and have not been associated, up until now, withmajor pathologies in man. Adenoviruses have thus been used to transfergenes of interest into the muscle (Ragot et al., 1993, Nature 361:647),the liver (Jaffe et al., 1992, Nature Genetics 1:372), the nervoussystem (Alki et al., 1993, Nature Genetics 3:224), tumors (Griscelli etal., 1998, PNAS 95:6367), intact or injured vascular endotheliums (vanBelle et al., 1998, Human Gene Therapy 9:1013), synovial tissue(Ghivizzani et al., 1998, PNAS 95:4613), and the like. Adenovirusvectors efficiently transfer genes to both replicating andnon-replicating cells (see, e.g., Crystal, 1995, Science 270:404-410).

Adenovirus Capsid

Characteristics of the adenovirus capsid are well known (see, e.g.,International Patent Publication WO 98/07877).

Various references describe the adenovirus hexon protein, and permitsome estimation of accessible sites. For example, Athapilly et al.(1994, J. Mol. Biol. 242:430-455) describes its refined crystalstructure at 2.9 A resolution. Crawford-Miksza et al. (1996, J. Virol.70: 1836-1844) reports the location and structure of seven hexon proteinhypervariable regions containing serotype-specific residues. Indeed,there has been a report of expression of a foreign epitope on thesurface of the adenovirus hexon (Crompton et al., 1994, J. Gen. Virol.75: 133-139). However, as shown in the examples, infra, the article byCrompton et al. does not teach a reproducible method of targetingadenovirus by modifying the hexon protein.

Additional references provide information about the fiber protein(Chroboczek et al., 1995, In Doerfler W. & P. Böhm (Eds.), The molecularrepertoire of adenoviruses, pp. 163-200, Springer-Verlag; Stewart andBurnett, ibid., pp. 25-38; Xia et al., 1995, ibid., pp. 39-46 Fender etal., 1995 Virol., 214:110-117; Hong and Engler, 1996, J. Virol., 70:7071-7078). Inhibition of cell adhesion to the virus by syntheticpeptides of fiber knob of human adenovirus serotypes 2 and 3 and virusneutralization by anti-peptide antibodies has been reported (Liebermannet al., 1996, Virus Research, 45:111-121). Xia et al. (1994, Structure,2:1259-1270) report the crystal structure of the receptor-binding domainof adenovirus type 5 fiber protein at 1.7 A resolution.

Adenoviruses are nonenveloped, regular icosahedrons of about 65 to 80 nmin diameter. The adenoviral capsid comprises 252 capsomers, of which 240are hexons and 12 are pentons. The hexons and pentons are derived fromthree different viral polypeptides (Maizel et al., 1968, Virology, 36:115-125; Weber et al., 1997, Virology: 76, 709-724. The Ad5 hexoncomprises three identical polypeptides of 967 amino acids each, namelypolypeptide II (Roberts et al., 1986, Science, 232: 1148-1151). Thepenton comprises a penton base, which provides a point of attachment tothe capsid, and a trimeric fiber protein, which is noncovalently boundto and projects from the penton base.

The fiber protein comprises three identical proteinaceous subunits ofpolypeptide IV (582 amino acids) and comprises a tail, a shaft and aknob (Devaux et al., 1990, J. Molec. Biol., 215: 567-588). The fibershaft comprises pseudorepeats of 15 amino acids, which are believed toform two alternating β-strands and α-bends (Green et al., 1983, EMBO J.,2: 1357-1365). The overall length of the fiber shaft and the number of15 amino acid repeats varies between adenoviral serotypes. For example,the Ad2 fiber shaft is 37 nm long and comprises 22 repeats, whereas theAd3 fiber is 11 nm long and comprises 6 repeats. Sequencing of over tenfiber proteins from different adenoviral serotypes has revealed agreater sequence diversity than that observed among other adenoviralproteins. For example, the knob regions of the fiber proteins from theclosely related Ad2 and Ad5 serotypes are only 63% similar at the aminoacid level (Chroboczek et al., 1992, Virology, 186: 280-285), whereastheir penton base sequences are 99% identical. Ad2 and Ad5 fiberproteins, however, both likely bind to the same cellular receptor, sincethey cross-block each other's binding. In contrast, Ad2 and Ad3 fibersare only 20% identical (Signas et al., 1985, J. Virol., 53:672-678), andbind to different receptors (Defer et al., 1990, J. Virol., 64(8),3661-3673).

Adenovirus serotype 2 has been shown to use the fiber and the pentonbase to interact with distinct cellular receptors to attach to andefficiently infect a cell (Wickham et al., 1993, Cell, 73: 309-319).First, the virus uses a receptor binding domain localized in the fiberknob (Henry et al., 1994, J. Virol., 68(8): 5239-5246) to attach to oneof at least two cell-surface receptors (Hong et al., 1997, EMBO J.,16:2294-2306; Bergelson et al., 1997, Science, 275:1320-1323; Phillipsonet al., 1968, J. Virol., 2: 1064-1075; Wickham et al., 1993 supra.;Svensson et al., 1981, J. Virol., 38: 70-81; and DiGuilmi et al., 1995,Virus Res., 38: 71-81). Then, following viral attachment, the pentonbase binds to a specific member of a family of heterodimericcell-surface receptors called integrins. For the Ad2 and Ad5 serotypes,which possess the long-shafted fibers, the penton base is notsignificantly involved in the initial viral attachment to host cells(Wickham et al., 1993, supra).

Most integrins recognize short linear stretches of amino acids in aligand, such as the tripeptide RGD, which is found in the majority ofextracellular matrix ligands. The integrin α_(IIb)β₃ binds fibrinogenvia the amino acid sequence KQAGD (SEQ ID NO:) (Kloczewiak et al., 1984,Biochemistry, 23, 1767-1774), and α₄β₁ binds fibronectin via the coresequence EILDV (SEQ ID NO:) (Komoriya et al., 1991, J. Biol. Chem., 266:15075-15079). Another structural motif, NPXY (SEQ ID NO:), which ispresent in the β subunits of α_(v)-containing integrins, also has beenshown to be important for integrin-mediated internalization (Suzuki etal., 1990, Proc. Natl. Acad. Sci. USA, 87: 5354).

Once Ad2 or Ad5 attaches to a cell via its fiber, it undergoesreceptor-mediated internalization into clathrin-coated endocyticvesicles by penton base binding to integrins. Ultimately, the viralparticles are transported to the nuclear pore complex of the cell, wherethe viral genome enters the nucleus, thereby initiating expression fromthe virus chromosome.

A drawback to the use of adenovirus in gene therapy, however, is thatall cells that comprise receptors for the adenoviral fiber and pentonbase will internalize the adenovirus, and, consequently, the gene(s)being administered—not just the cells in need of therapeutic treatment.Also, cells that lack either the fiber receptor or the penton basereceptor will be impaired in adenoviral-mediated gene delivery. Cellsthat appear to lack an adenoviral fiber receptor, are transduced byadenovirus, if at all, with a very low efficiency (Curiel et al., 1992,supra; Cotton et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 4033-4037;Wattel et al., 1996, Leukemia, 10: 171-174). Accordingly, effectivelydirecting adenoviral entry to specific cells and in some cases expandingthe repertoire of cells amenable to adenovirus-mediated gene therapy,represents an important goal to improve current vectors. Both approachesalso could potentially reduce the amount of adenoviral vector that isnecessary to obtain gene expression in the targeted cells and, thus,potentially reduce side effects and complications associated with higherdoses of adenovirus.

Adenovirus Targeting Strategies

Various strategies have been employed to modify adenovirus tropism,i.e., to target adenovirus to specific cell types not normally infectedefficiently by wild-type adenovirus vectors (see, e.g., InternationalPatent Publication WO 98/07877). Fiber protein, hexon protein, andpenton base protein modification strategies have been employed.

U.S. Pat. No. 5,559,099 describes a recombinant virus comprising achimeric penton base protein with a nonpenton amino acid sequencespecific for a receptor, antibody, or epitope in addition to or in placeof a wild-type penton base sequence, and a therapeutic gene.

U.S. Pat. No. 5,543,328 claims an adenovirus wherein the adenovirusfiber includes a ligand specific for a receptor located on a desiredcell type. Such an adenovirus fiber can be prepared by removing all or aportion of the fiber protein head portion and replacing it with ligand,or by creating a fusion between a full length fiber protein and aligand.

International Patent Publication WO 95/26412 discloses modification ofadenovirus full length fiber protein to contain a C-terminal linker forattachment of a ligand. Inclusion of a linker is stated to avoid stericinterference with formation of a fiber protein homotrimer.

International Patent Publication WO 96/26281 discloses a recombinantadenovirus comprising (a) a chimeric fiber protein with a normativeamino acid sequence in addition to or in place of a native fibersequence, (b) a therapeutic gene, and, optionally, (c) a normativetrimerization domain in place of the native fiber amino acidtrimerization domain. The normative amino acid sequence can be a proteinbinding sequence, and may be located at the C-terminus.

International Patent Publication WO 97/20051 discloses a chimericadenovirus coat protein with a normative amino acid sequence that isable to direct vector entry into cells more efficiently than a vectorwith wild-type coat protein. The normative sequence can be inserted intoor in place of an internal coat protein sequence, at or near theC-terminus of the coat protein, or in an exposed loop of the coatprotein. The coat protein can be a fiber protein, a penton base protein,or a hexon protein. A spacer sequence can be included.

Other targeting techniques rely on post-expression modification of theviral coat protein, e.g., by covalent or non-covalent binding ofbridging targeting groups or targeting groups (see, e.g., InternationalPatent Application No. WO 97/05266; International Patent Publication WO97/23608; International Patent Publication WO 97/32026).

Despite these efforts, there remains a need in the art to identifysuitable modes of insertion of the binding peptide so that it isaccurately displayed at the capsid surface to allow virus growth andspecific binding to its cognate receptor(s).

There is a further need to define critical parameters that permitrecognition of such sequences. Yet another need in the art is to providespecific targeting peptide sequences suitable for directing adenovirusvectors to target cells in vivo. These and other needs of the art areaddressed by the present invention.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication. Each of the references disclosed herein is incorporated byreference in the application in its entirety.

SUMMARY OF THE INVENTION

The present invention advantageously provides an effective targetedadenovirus vector. The targeted vector of the invention is characterizedby an appropriate deletion of amino acids from an effective site ineither the hexon protein or the fiber protein.

Thus, in a more specific embodiment the invention relates to anadenovirus from which at least a part of the hexon HRV5 loop is replacedwith a binding peptide, or targeting sequence, flanked by connectingamino acid spacers so as to functionnaly display its binding specificityat the capsid surface. In a specific embodiment the adenovirus comprisesa deletion of about 6 to 17 amino acids from the hexon HVR5 looppreferably not exceeding 14 amino acids.

In an other specific embodiment the invention relates to an adenovirusfrom which at least a part of the fiber HI loop is replaced with abinding peptide, or targeting sequence, flanked by connecting amino acidspacers so as to functionnaly display its binding specificity at thecapsid surface. In a specific embodiment the adenovirus comprises adeletion of about 6 to 17 amino acids from the hexon HI loop preferablynot exceeding 11 amino acids.

In a further embodiment the invention relates to a recombinantadenovirus vector wherein a binding peptide, or targeting sequence, isconnected to the C-terminus of the fiber by a connecting spacer, orlinker, so as to functionaly display its binding specificity at thecapsid surface.

In a more specific embodiment, about 13 amino acids are deleted from thehexon HVR5 loop corresponding to about amino acid residue 269 to aboutamino acid residue 281 of adenovirus serotype 5 (Ad5). In anotherspecific embodiment, about 11 amino acids are deleted from the fiberprotein HI loop corresponding to about amino acid residue 538 to aboutamino acid residue 548 of adenovirus serotype 5 (Ad5). A targetingpeptide is inserted at the site of the deletion. A particular advantageof the invention is the discovery that the targeting peptide sequenceshould be connected by a first spacer at the N-terminus and a secondspacer at the C-terminus of the targeting sequence, wherein the spacerscomprise a flexible amino acid residue. Preferably, the first spacer orthe second spacer, or both, comprises an amino acid selected from thegroup consisting of glycine and serine.

In a specific aspect, the targeted adenovirus modified in the hexonprotein advantageously employs dipeptide spacers consisting of flexibleamino acid residues. In a specific embodiment, the first and secondspacers are a Gly-Ser dipeptide. In a further specific embodiment ofthis aspect, the first spacer is a glycine residue.

In an aspect of the invention in which the fiber protein HI loop ismodified, the first and second spacers are advantageously tri-peptidesconsisting of flexible amino acid residues. In a specific embodiment ofthis aspect of the invention, the first and second spacers are aGly-Ser-Ser tri-peptide.

Preferably, the targeting sequence is a ligand epitope for aurokinase-type plasminogen activator receptor (UPAR). In particular, thetargeting sequence can be selected from the group consisting ofLNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2);AEPMPHSLNFSQYLWT (SEQ ID NO:3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4);RGHSRGRNQNSR (SEQ ID NO: 5); and NQNSRRPSRA (SEQ ID NO: 6).

In an alternative embodiment in which the hexon protein is modified, thetargeting sequence including the spacers is selected from the groupconsisting of: gly-ser-LNGGTCVSNKYFSNIHWCN-gly-ser; (SEQ ID NO:7)gly-ser-LNGGTAVSNKYFSNIHWCN-gly-ser; (SEQ ID NO:8)gly-ser-AEPMPHSLNFSQYLWT-gly-ser; (SEQ ID NO:9)gly-ser-AEPMPHSLNFSQYLWYT-gly-ser; (SEQ ID NO:10)gly-ser-RGHSRGRNQNSR-gly-ser; (SEQ ID NO:11) gly-ser-NQNSRRPSRA-gly-ser;(SEQ ID NO:12) gly-ser-CDCRGDCFC-gly-ser; (SEQ ID NO:13)gly-ser-DCRGDCF-gly-ser; (SEQ ID NO:14) and gly-ser-KKKKKKK-gly-ser.(SEQ ID NO:15)

In an alternative embodiment in which the fiber protein is modified, thetargeting sequence including the spacers is selected from the groupconsisting of: (SEQ ID NO:16 )gly-ser-ser-LNGGTCVSNKYFSNIHWCN-gly-ser-ser; (SEQ ID NO:17 )gly-ser-ser-LNGGTAVSNKYFSNIHWCN-gly-ser-ser; (SEQ ID NO:18 )gly-ser-ser-AEPMPHSLNFSQYLWT-gly-ser-ser; (SEQ ID NO: 19 )gly-ser-ser-AEPMPIISLNFSQYLWYT-gly-ser-ser; (SEQ ID NO:20 )gly-ser-ser-RGHSRGRNQNSR-gly-ser-ser; (SEQ ID NO:21:)gly-ser-ser-NQNSRRPSRA-gly-ser-ser; (SEQ ID NO:22 )gly-ser-ser-CDCRGDCFC-gly-ser-ser; (SEQ ID NO:23 )gly-ser-ser-DCRGDCF-gly-ser-ser; (SEQ ID NO:24 )gly-ser-ser-KKKKKKK-gly-ser-ser (SEQ ID NO: 143)ser-ser-RGHSRGRNQNSRRPSRA-gly-ser; (SEQ ID NO: 144)tyr-ser-glu-RGFISRGRNQNSR-gly-ser; (SEQ ID NO: 145)tyr-gln-glu-RGHSRGRNQNSR-gly-ser; (SEQ ID NO: 146)ser-ser-ser-RGHSRGRNQNSR-gly-ser; and (SEQ ID NO: 147)ser-ser-RGHSRGRNQNSR-gly-gly.

Preferably the connecting spacer or linker comprises an amino acidselected from the group consisting of glycine, serine, threonine,alanine, cysteine, aspartate, asparagine, methionine and proline. In arefered embodiment the first amino acid in the spacer is a proline.

The recombinant adenovirus can be derived from a human adenovirusserotype, in particular from human adenovirus subgroup C, such as humanadenovirus serotype 5.

The fiber protein can be modified to have a fiber shaft that is shorterthan a wild-type fiber shaft, in particular by an in-frame deletion orby replacing it with the shaft from another serotype. The fiber shaftcan be from subgroup C and comprises an in-frame deletion encompassingrepeats 4 to 16 or repeats 4 to 19 or from subgroup C and has beenshortened by replacing it with the shaft from serotype 3 (Ad3)

According to either aspect of the invention (modification of the hexonor the fiber), the fiber protein can be modified to be shorter than inthe wild-type sequence. For example, the fiber protein can be modifiedto contain only repeats 1 to 3 and 17 to 22 of Ad5; repeats 1 to 3 and20 to 22 of Ad5; or an adenovirus serotype 3 (Ad3) shaft in place of theendogenous Ad5 shaft.

In a specific embodiment, exemplified infra, the adenovirus is aserotype 5 adenovirus.

In a further embodiment, the present invention provides a specifictargeted adenovirus vector comprising a linker peptide and a targetingpeptide at the C-terminus of the fiber protein.

Preferably, the targeting sequence is a ligand for a UPAR, such as CD87,a peptide fragment from FGF-1 binding to heparin, comprising between 7and 15 amino acids, is composed of 5 to 10 lysine residues, preferablyof almost 7 lysine residues, or is composed of between 5 and 10 Arg-Argand Leu-Leu motifs.

Preferably, the targeting sequence is selected from the group consistingof LNGGTCVSNKYFSNIHWCN; (SEQ ID NO: 1) LNGGTAVSNKYFSNIHWCN; (SEQ ID NO:2) AEPMPHSLNFSQYLWYT; (SEQ ID NO: 3) AEPMPHSLNFSQYLWYT; (SEQ ID NO: 4)RGHSRGRNQNSR; (SEQ ID NO: 5) NQNSRRPSRA; (SEQ ID NO: 6) RRLLRRLLRR; (SEQID NO: 133) and KRGPRTHYGQK; (SEQ ID NO: 134)

Preferably the linker peptide comprises the sequence PKRARPGS (SEQ IDNO.149) and the targeting sequence including the linker peptidecomprises the sequences PKRARPGSKKKKKKK (SEQ ID NO.132),PKRARPGSRRLLRRLLRR (SEQ ID NO.141) or PKRARPGSKRGPRTHYGQK (SEQ IDNO.140).

Naturally, given the targeted adenoviruses disclosed above and ingreater detail herein, the present invention provides a method formodifying the cellular tropism of an adenovirus vector, comprisingdeleting a native amino acid sequence from a site in a capsid protein ofthe adenovirus; and inserting a targeting peptide sequence connected bya first spacer at the N-terminus and a second spacer at the C-terminusof the targeting sequence, wherein the spacers comprise a flexible aminoacid residue. According to this aspect of the invention, the targetingpeptide is inserted in a deletion site selected from the groupconsisting of about 13 amino acids from the hexon HVR5 loopcorresponding to about amino acid residue 269 to about amino acidresidue 281 of adenovirus Ad5; and about 11 amino acids from the fiberprotein HI loop corresponding to about amino acid residue 538 to aboutamino acid residue 548 of Ad5. In a preferred embodiment, the firstspacer comprises an amino acid selected from the group consisting ofglycine and serine. In another preferred embodiment, the second spacercomprises an amino acid selected from the group consisting of glycineand serine.

Are also encompassed by the present invention:

-   -   an adenovirus hexon comprising a deletion of about 13 amino        acids from the HVR5 loop corresponding to about amino acid        residue 269 to about amino acid residue 281 of adenovirus        serotype 5 (Ad5) and an insertion at the site of the deletion of        a targeting peptide sequence connected by a first spacer at the        N-terminus and a second spacer at the C-terminus of the        targeting sequence, wherein the first and second spacers        comprise a flexible amino acid residue.    -   an adenovirus fiber protein comprising a deletion of about 11        amino acids from the HI loop corresponding to about amino acid        residue 538 to about amino acid residue 548 of adenovirus        serotype 5 (Ad5) and an insertion at the site of the deletion of        a targeting peptide sequence connected by a first spacer at the        N-terminus and a second spacer at the C-terminus of the        targeting sequence, wherein the first and second spacers        comprise a flexible amino acid residue.    -   an adenovirus fiber protein comprises a linker peptide and a        targeting peptide at its C-terminus.

The invention further specifically provides a method for targeting cellsthat express a urokinase-type plasminogen activator receptor (UPAR). Inparticular, this method comprises using adenoviruses modified to exposeat the capsid surface the specific UPAR targeting peptides disclosedabove. Alternatively, the invention provides for modifying the hexonHVR5 loop or the fiber protein HI loop by inserting the specificsequences, including spacer groups, defined above.

The method for targeting a specific cell type in accordance with theinvention can be further enhanced by shortening the fiber protein shaft,e.g., such that the fiber shaft only contains repeats 1 to 3 and 17 to22 of Ad5; repeats 1 to 3 and 20 to 22 of Ad5; or with an Ad3 shaft.Repeats are as described by Chroboczek et al (1995, Current Topics inMicrobiology and Immunology, Springer Verlag, 199 :163-200).

The invention further provides a method for preferentially expressing agene in a target cell comprising contacting a population of cellscontaining the target cell with the targeted adenovirus vector of theinvention, wherein the targeting sequence is a ligand epitope for areceptor on the target cell. In particular, the invention provides amethod for preferentially expressing a gene in a target cell thatexpresses a UPAR comprising contacting a population of cells containingthe target cell with the targeted adenovirus vectors of the inventionthat are modified to display a UPAR binding peptide. In this embodiment,the targeted adenovirus vector preferably comprises a heterologoustherapeutic gene or nucleic acid for transduction of actively dividingand/or motile cells, including tumor cells and its metastases, tumorvasculature, activated endothelial cells, activated smooth muscle cells. . . However, UPAR targeted vectors can also be considered as candidatevectors for transduction of other tissues as muscle, brain, heart, etc.More particularly, the nucleic acid encodes a therapeutic polypeptidewhich acts as an angiogenesis inhibitor, an angiogenic factor, aconditional suicide effector, a tumor suppressor, a growth-arrestprotein (GAX), or any secreted polypeptide.

In particular, the invention provides a method for preferentiallyexpressing a gene in a target cell that expresses an integrin comprisingcontacting a population of cells containing the target cell with thetargeted adenovirus vector of the invention that are modified to displayan integrin binding peptide. In this embodiment, the targeted adenovirusvector preferably comprises a therapeutic gene or nucleic acid fortransduction of actively dividing and/or motile cells, including tumorcells and its metastases, tumor vasculature, activated endothelialcells, activated smooth muscle cells . . . However, integrin targetedvectors can also be considered as candidate vectors for transduction ofother tissues as skeletal muscle, brain, heart, hematopoietic cells,ischemic tissues, etc. More particularly, the nucleic acid encodes atherapeutic polypeptide which acts as an angiogenesis inhibitor, anangiogenic factor, a conditional suicide effector, a tumor suppressor, agrowth-arrest protein (GAX), a cell survival-promoting factor (inparticular members of the Akt/PKB family) or any secreted polypeptide.

Thus a further object of the present invention is a method for thetreatment of a disease by gene therapy comprising the step ofadministering a targeted adenovirus vector as disclosed hereabove, orobtained according to the method disclosed hereabove.

Still an object of the present invention is a medecine containing atargeted adenovirus vector as disclosed hereabove, or obtained accordingto the method as disclosed hereabove and the use of such a targetedadenovirus vector for manufacturing a medecine for the treatment of adisease by gene therapy. A further object of the present invention is apharmaceutical composition containing such targeted adenovirus vectorsand a efficient quantity of a pharmaceutically active excipient.

Thus, an object of the invention is to provide for tropism-modifiedadenovirus vectors without adversely impacting productivity of thevectors.

A further object of the invention is to identify suitable mode ofinsertion so that the binding peptide is accessible and effectivelyrecognizes its specific receptors.

Still another object of the invention is to identify the number of aminoacid residues that can be deleted from the native adenovirus capsidprotein so that the targeting peptide sequence can be effectivelyinserted.

Yet another object of the invention is to provide the effective size andcharacteristics of spacer sequences to be included at the ends of theinserted targeting peptide to permit adoption by the targeting peptideof an accessible conformation that permits effective binding to thetarget receptor.

These and other objects of the invention are further provided by theaccompanying Drawings and the following Detailed Description of theInvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Neutralization assay on W162 cells with virus modified in thehexon HVR5 region.

FIG. 2. Structure of the shortened fiber construct in which theadenovirus serotype 3 (Ad3) shaft is inserted in place of the Ad5 shaft.

FIG. 2A: General description of the hybrid Ad3/5 fiber;

FIG. 2B: Detailed description of the hybrid Ad3/5 fiber

FIG. 3: Knob competition in 293 cells.

FIG. 4: Infection of hSMC with various viruses in presence of increasingdoses of soluble heparin.

FIG. 5: Infection of hSMC with viruses preincubated with increasingdoses of soluble uPAR.

FIGS. 6 and 7: Gax expression in human SMC infected with targetedadenovirus.

FIGS. 8A to 8C and 9: Infection of Hs578T with different targetedviruses.

FIG. 10: Infection of Hs578T with virus AE43 preincubated withincreasing doses of soluble uPAR.

FIG. 11: Infection of Hs578T with virus AE43 preincubated withincreasing doses of soluble uPAR or soluble knob.

FIG. 12: Infection of Hs578T with Vn4 containing viruses.

FIG. 13: Infection of NIH-3T3 with a large range of targeted viruses.

FIGS. 14A and 14B: Infection of Hs578T with virus viruses BC15X (A) andAE43 (B) preincubated with increasing doses of soluble uPAR.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, there has been keen interest in the art to modifyadenovirus tropism so as to permit targeting adenovirus vectors tospecific target cells, including those cells that are not efficientlyinfected by adenoviruses. While there have been some successes inachieving this goal, the present inventors recognized that a practicalsolution to this problem, i.e., a solution that would not adverselyaffect viral productivity and that would permit a satisfactory increasein cell specificity, had not been achieved. In particular, optimum sitesfor incorporation of a targeting peptide in a viral capsid protein, thesize of a deletion from the native capsid protein (if any), the size ofan inserted targeting sequence, and the presence and nature of anylinker sequences joining the targeting sequence to the capsid protein,are not described in the prior art. The present invention advantageouslyaddresses these issues, and provides highly effective targeting by:providing optimized sites for insertion of a targeting sequence,including the size of the native sequence to be deleted to make room forthe targeting peptide; identifying an appropriate size for the targetingsequence; and disclosing the size and characteristics of linkers thatpermit accessibility and specific recognition of the targeting peptide;and identifying interesting cell marker as a target receptor.

In particular, the present inventors set out to introduce an accessibleforeign peptide on the surface of the adenoviral capsid, and thus tomodify the natural tropism of the virus. With this in mind, a series ofconstructs with modified hexons or fibers incorporating a neutralizingepitope from poliovirus type 1 were designed. The poliovirus sequencewas chosen as cognate neutralizing antibodies were readily available todocument in great details its accessibility and functionality.

Modification of the hexon and fiber to restrict infection to specifictarget cells requires that the adenovirus fiber not be able toefficiently interact with its native cellular receptors In the case ofhexon modified capsid, it also requires that the binding peptide caninteract directly with its cognate receptor on the cell surface withoutsteric hindrance from the fiber. For these purposes, the possibility ofshortening the shaft of the fiber was investigated.

The invention is based, in part, on experiments that identified sitessuitable for insertion of functional targeting sequences in the hexonprotein and in the fiber protein. It was discovered that replacement ofa portion of the hexon HVR5 loop and fiber HI loop permitted insertionof targeting sequences without adversely affecting viral productivity.Furthermore, the data showed that incorporation of flexible linkerpeptides at the ends of the inserted sequences was critical toaccessibility or recognition of the targeting sequence. Moreover,incorporation of ligands specific for uPAR or for some integrins wassuccessfully achieved in terms of accessibility of the ligand,productivity of the modified viruses, transduction efficiency of thetarget cell types as shown in in vitro and in vivo experiments. Finally,it was also shown that shortening of the fiber efficiently reduces virusaffinity for natural host cell, which makes this strategy attractive forablation of natural tropism of Ad5.

In a preferred embodiment, the different approaches are combined.Preferably, the fiber protein is shortened in a virus having a modifiedhexon HVR5 loop or fiber HI loop. In a further example, a virus with ashortened fiber could have an insertion in HVR5 or in HI to target UPARas the primary step of the infection, and an insertion in HI loop or inHVR5 (respectively) to target an integrin to favor the internalizationof the virus in the endosome. Any suitable membrane receptor can also betargeted using the same strategy, i.e. incorporation of high affinityligands in the hexon and/or the fiber in combination with shortening ofthe fiber.

The Detailed Description of the Invention is further elaborated insections relating to specific definitions, adenovirus vectors, targetingpeptide sequences, and uses of the targeted adenovirus vectors. Thevarious headers and organization of the sections are provided for thesake of clarity and convenience, and are not in any way to be deemedlimiting.

Definitions

Various terms are used throughout the specification and claims. Wherenot otherwise defined, the following definitions apply:

As used in the art, a <<vector>> is any means for the transfer of anucleic acid according to the invention into a host cell. For purposesof the present invention, the term vector is used to modify “adenovirus”so as to reflect that the adenovirus has been genetically engineered totransfer a nucleic acid of interest (a gene under control of, oroperably linked to, an expression control sequence) into the targetcell. Adenovirus vectors of the invention are described in greaterdetail, infra.

A cell has been “transfected” or “infected” by an adenovirus vector ofthe invention when viral DNA has been introduced inside the cell. A cellhas been “transformed” by exogenous or heterologous DNA when thetransfected DNA effects a phenotypic change.

The term “corresponding to” is used herein to refer similar orhomologous sequences, whether the exact position is identical ordifferent from the molecule to which the similarity or homology ismeasured. A nucleic acid or amino acid sequence alignment may includespaces. Thus, the term “corresponding to” refers to the sequencesimilarity, and not the numbering of the amino acid residues ornucleotide bases. Examples include hexon proteins or fiber proteins fromother adenovirus serotypes (2, 3, etc.) besides the type 5 adenovirus(Ad5) exemplified herein. Those of ordinary skill in the art arefamiliar with homologous adenovirus capsid proteins.

In other words, the present invention provides for modification ofhomologous capsid proteins from other adenovirus species using theoptimized parameters defined herein for Ad5. As used herein, the term“homologous” in all its grammatical forms and spelling variations refersto the relationship between proteins that possess a “common evolutionaryorigin,” including proteins from superfamilies (e.g., the immunoglobulinsuperfamily) and homologous proteins from different species (e.g.,myosin light chain, etc.) (Reeck et al., 1987, Cell 50:667). Suchproteins (and their encoding genes) have sequence homology, as reflectedby their high degree of sequence similarity.

The term “deletion” refers to the removal of native amino acid residuesfrom a defined region of an adenovirus capsid protein, i.e., a hexon orfiber protein. According to the invention, a preferred size for such adeletion is between about 10 and about 20 amino acids. More preferably,the size of the deletion is between about 10 and about 15 amino acids.In specific embodiments, 11 and 13 amino acid sequences were deleted.

The term “spacer” or “spacer peptide” or <<linker>> or <<linkerpeptide>> is used herein to refer to a sequence of about one to aboutthree amino acids that is included to connect the binding peptide to itscapsid carrier protein. The spacer or the linker is preferably made upof amino acid residues with high degrees of freedom of rotation, whichpermits the targeting peptide to adopt a conformation that is recognizedby its binding partner (e.g., receptor). Preferably no more than threeamino acids are included in the spacer; more preferably, the spacerconsists of two amino acids. Preferred amino acids for the spacer areglycine and serine. In specific embodiments, the spacer is a peptidehaving the sequence Gly-Ser or Gly-Ser-Ser.

As used herein, the term “about” or “approximately” means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange.

Adenovirus Vectors

Adenoviruses are eukaryotic DNA viruses that can be modified toefficiently deliver a nucleic acid of the invention to a variety of celltypes. Various serotypes of adenovirus exist. Of these serotypes,preference is given, within the scope of the present invention, to usingtype 2 or type 5 human adenoviruses (Ad 2 or Ad 5) or adenoviruses ofanimal origin (see WO94/26914). Those adenoviruses of animal originwhich can be used within the scope of the present invention includeadenoviruses of canine, bovine, murine (example: Mav1, Beard et al.,Virology 75 (1990) 81), ovine, porcine, avian, and simian (example: SAV)origin. Preferably, the adenovirus of animal origin is a canineadenovirus, more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61strain (ATCC VR-800)).

Adenoviral vectors are commonly used for in vitro, in vivo or ex vivotransfection and gene therapy procedures. Preferably, the adenoviralvectors are replication defective, that is, they are unable to replicateautonomously in the target cell. In general, the genome of thereplication defective viral vectors within the scope of the presentinvention lack at least one region which is necessary for thereplication of the virus in the infected cell. These regions can eitherbe eliminated (in whole or in part), be rendered non-functional by anytechnique known to a person skilled in the art. These techniques includethe total removal, substitution (by other sequences, in particular bythe inserted nucleic acid), partial deletion or addition of one or morebases to a region required for virus propagation. Such techniques may beperformed in vitro (on the isolated DNA) or in situ, using thetechniques of genetic manipulation or by treatment with mutagenicagents. For purposes of the present invention, the replication defectivevirus retains the sequences of its genome which are necessary forencapsidating the viral particles. Defective viruses, which entirely oralmost entirely lack viral genes, may also be used.

The replication defective adenoviral vectors of the invention compriseat least the ITRs, an encapsidation sequence and the nucleic acid ofinterest. Preferably, at least the E1 region of the adenoviral vector isrendered non-functional. The deletion in the E1 region preferablyextends from nucleotides 455 to 3329 in the sequence of the Ad5adenovirus (PvuII-BglII fragment) or 382 to 3446 (HinfII-Sau3Afragment), or 382-3512 (HinfI-RsaI fragment). Other regions may also bemodified, in particular the E3 region (WO95/02697), the E2 region(WO94/28938), the E4 region (WO94/28152, WO94/12649 and WO95/02697), theIVa2 region (WO96/10088) or in any of the late genes L1-L5.

In a preferred embodiment, the adenoviral vector has a deletion in theE1 region (Ad 1.0). Examples of E1-deleted adenoviruses are disclosed inEP 185,573, and in FR 97/14383, filed 11 Nov. 1997, the contents ofwhich are incorporated herein by reference. In another preferredembodiment, the adenoviral vector has a deletion in the E1 and E4regions (Ad 3.0). Examples of E1/E4-deleted adenoviruses are disclosedin WO95/02697 and WO96/22378, the contents of which are incorporatedherein by reference. In still another preferred embodiment, theadenoviral vector has a deletion in the E1 region into which an E4functional region and the nucleic acid sequence are inserted (see FR9413355, the contents of which are incorporated herein by reference).Another adenovirus vector for use in the invention is described in WO96/10088.

The replication defective recombinant adenoviruses according to theinvention can be prepared by any technique known to the person skilledin the art (Levrero et al., 1991, Gene 101: 195; EP 185 573; Graham,1984, EMBO J. 3: 2917). In particular, they can be prepared byhomologous recombination between an adenovirus and a plasmid whichcarries, inter alia, the DNA sequence of interest. The homologousrecombination is effected following cotransfection of the saidadenovirus and plasmid into an appropriate cell line. The cell linewhich is employed should preferably (i) be transformable by the saidelements, and (ii) contain the sequences which are able to complementthe part of the genome of the replication defective adenovirus,preferably in integrated form in order to avoid the risks ofrecombination. Examples of cell lines which may be used are the humanembryonic kidney cell line 293 (Graham et al., 1977, J. Gen. Virol. 36:59) which contains the left-hand portion of the genome of an Ad5adenovirus (12%) integrated into its genome, PER.C6 (Bout et al., 1997,Cancer Gene Therapy 4:324; Fallaux et al, 1998, Hum Gen Ther 9:1909-1917), and cell lines which are able to complement the E1 and E4functions, as described in applications WO94/26914 and WO95/02697. In apreferred embodiment, an E. coli vector system is used to generate theadenovirus backbone (International Patent Publication WO 96/25506;Crouzet et al. 1997, Proc. Natl. Acad. Sci. 94:1414-1419).

Indeed, the general techniques for preparing adenoviruses of theinvention are well known in the art. Such techniques are explained fullyin the literature. See, e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, Second Edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al.,1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N.Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);Nucleic Acid Hybridization [B. D. Hames & S. J. ÊHiggins eds. (1985)];Transcription And Translation [B. D. Hames & S. J. Higgins, eds.(1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; ImmobilizedCells And Enzymes [IRL Press, (1986)]; B. ÊPerbal, A Practical Guide ToMolecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, Inc. (1994). Incorporation ofcassette insertion sites, whether for insertion of a heterologous geneor for insertion of the targeting sequence in the hexon or fiber protein(as exemplified infra) facilitates such genetic manipulations. A“cassette” refers to a segment of DNA that can be inserted into a vectorat specific restriction sites. The segment of DNA encodes a polypeptideof interest, and the cassette and restriction sites are designed toensure insertion of the cassette in the proper reading frame fortranscription and translation.

Recombinant adenoviruses are recovered and purified using standardmolecular techniques, which are well known to one of ordinary skill inthe art (see, e.g., International Patent Publication WO 98/00524,International Patent Publicaiton WO 96/27677; and International PatentPublication WO 97/08298).

Expression of heterologous genes by adenovirus vectors of the invention.The adenovirus vectors of the invention preferably contain a DNA codingsequence for a heterologous gene. A DNA “coding sequence” is adouble-stranded DNA sequence which is transcribed and translated into apolypeptide in a cell in vitro or in vivo when placed under the controlof appropriate regulatory sequences. The boundaries of the codingsequence are determined by a start codon at the 5′ (amino) terminus anda translation stop codon at the 3′ (carboxyl) terminus. A codingsequence can include, but is not limited to, prokaryotic sequences, cDNAfrom eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g.,mammalian) DNA, and even synthetic DNA sequences. If the coding sequenceis intended for expression in a eukaryotic cell, a polyadenylationsignal and transcription termination sequence will usually be located 3′to the coding sequence. A coding sequence is “under the control” oftranscriptional and translational control sequences in a cell when RNApolymerase transcribes the coding sequence into mRNA, which is thentrans-RNA spliced and translated into the protein encoded by the codingsequence. In another embodiment, the nucleic acid of interest is nottranslated into a polypeptide but acts as a specific anti-sensetherapeutic molecule, or as a therapeutic decoy.

Transcriptional and translational control sequences are DNA regulatorysequences, such as promoters, enhancers, terminators, and the like, thatprovide for the expression of a coding sequence in a host cell. Ineukaryotic cells, polyadenylation signals are control sequences. A“promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site (convenientlydefined for example, by mapping with nuclease S1), as well as proteinbinding domains (consensus sequences) responsible for the binding of RNApolymerase.

Expression of a heterologous protein may be controlled by anypromoter/enhancer element known in the art, but these regulatoryelements must be functional in the target cell selected for expression.Promoters which may be used to control gene expression include, but arenot limited to, the cytomegalovirus immediate early (CMV-IE or CMV)promoter, the SV40 early promoter region (Benoist and Chambon, 1981,Nature 290:304-310), the promoter contained in the 3′ long terminalrepeat of Rous sarcoma virus (Yamamoto, et al., 1980, Cell 22:787-797),the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1441-1445), the regulatory sequences of themetallothionein gene (Brinster et al., 1982, Nature 296:39-42); and theanimal transcriptional control regions, which exhibit tissue specificityand have been utilized in transgenic animals: elastase I gene controlregion which is active in pancreatic acinar cells (Swift et al., 1984,Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant.Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin genecontrol region which is active in pancreatic beta cells (Hanahan, 1985,Nature 315:115-122), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames etal., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in embryonic liver and hepatomas(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al.,1987, Science 235:53-58), alpha 1-antitrypsin gene control region whichis active in the liver (Kelsey et al., 1987, Genes and Devel.1:161-171), beta-globin gene control region which is active in myeloidcells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986,Cell 46:89-94), myelin basic protein gene control region which is activein oligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-712), myosin light chain-2 gene control region which is active inskeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

A “signal sequence” is included at the beginning of the coding sequenceof a protein to be exported at the cell surface, or secreted. Thissequence encodes a signal peptide, N-terminal to the mature polypeptide,that directs the host cell to translocate the polypeptide into thesecretion pathway/compartment. The term “translocation signal sequence”is used herein to refer to this sort of signal sequence. Translocationsignal sequences can be found associated with a variety of proteinsnative to eukaryotes and prokaryotes, and are often functional in bothtypes of organisms.

Specific heterologous genes are discussed in the section relating touses of the vectors of the invention, infra.

Targeting Peptide Sequences

Any known targeting sequence can be incorporated in a hexon HVR5 loop orfiber protein in accordance with the present invention. Examples oftargeting peptides are ample in the literature. In general, any peptideligand can provide a targeting sequence based on the receptor-bindingsequence of the ligand. In immunology, such a sequence is referred to asan epitope, and the term epitope may be used herein to refer to thesequence of a ligand recognized by a receptor. Specifically, the term“ligand epitope of a receptor” refers to the sequence of a protein orpeptide that is recognized by a binding partner on the surface of atarget cell, which for the sake of convenience is termed a receptor.However, it should be understood that for purposes of the presentinvention, the term “receptor” encompasses signal-transducing receptors(e.g., receptors for hormones steroids, cytokines, insulin, and othergrowth factors), recognition molecules (e.g., MHC molecules, B- orT-cell receptors), nutrient uptake receptors (such as transferrinreceptor), lectins, ion channels, adhesion molecules, extracellularmatrix binding proteins, and the like that are located and accessible atthe surface of the target cell. Targeting peptides of the invention canbind to polypeptide or carbohydrate moieties on such receptors.

The size of the targeting peptide can vary within certain parameters. Asshown in the examples, inserting peptides longer than the deletedsequence did not adversely impact viral productivity. Thus, theinvention contemplates using a targeting sequence that is several foldlonger than the deleted segment; preferably, the inserted peptide is nomore than 200% longer than the deleted segment, and more preferably theinserted peptide is about the same size as the deleted segment.

Due to the degeneracy of nucleotide coding sequences, other DNAsequences which encode substantially the same amino acid sequence as atargeting peptide sequence may be used in the practice of the presentinvention. These include but are not limited to allelic variants,homologous variants from other species, variants in which a conservativeamino acid substitution is effected, and variants in which a highlypolymorphic amino acid residue (which is presumably not important forbinding specificity) is changed. For example, one or more amino acidresidues within the sequence can be substituted by another amino acid ofa similar polarity, which acts as a functional equivalent, resulting ina silent alteration. Substitutes for an amino acid within the sequencemay be selected from other members of the class to which the amino acidbelongs. For example, the nonpolar (hydrophobic) amino acids includealanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophanand methionine. Amino acids containing aromatic ring structures arephenylalanine, tryptophan, and tyrosine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Such alterations will not beexpected to significantly affect apparent molecular weight as determinedby polyacrylamide gel electrophoresis, or isoelectric point.

Particularly preferred substitutions are:

-   -   Lys for Arg and vice versa such that a positive charge may be        maintained;    -   Glu for Asp and vice versa such that a negative charge may be        maintained;    -   Ser for Thr such that a free —OH can be maintained; and    -   Gln for Asn such that a free CONH₂ can be maintained.

Such variants may be encoded by highly similar nucleic acids. Suchnucleic acids will, in general, hybridize to a nucleic acid (includingan oligonucleotide probe) that encodes the native amino acid sequence.As demonstrated in the examples, infra, various modifications can beintroduced by single base changes that do not affect hybridization (ofPCR primers), but that can create an endonuclease cleavage site or aaltered amino acid residue.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other nucleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al., supra). The conditions oftemperature and ionic strength determine the “stringency” of thehybridization. For preliminary screening for homologous nucleic acids,low stringency hybridization conditions, corresponding to a T_(m) of 55°C., can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or30% formamide, 5×SSC, 0.5% SDS). Moderate stringency hybridizationconditions correspond to a higher T_(m), e.g., 40% formamide, with 5× or6×SCC. High stringency hybridization conditions correspond to thehighest T_(m), e.g., 50% formamide, 5× or 6×SCC. Hybridization requiresthat the two nucleic acids contain complementary sequences, althoughdepending on the stringency of the hybridization, mismatches betweenbases are possible. The appropriate stringency for hybridizing nucleicacids depends on the length of the nucleic acids and the degree ofcomplementation, variables well known in the art. The greater the degreeof similarity or homology between two nucleotide sequences, the greaterthe value of T_(m) for hybrids of nucleic acids having those sequences.The relative stability (corresponding to higher T_(m)) of nucleic acidhybridizations decreases in the following order: RNA:RNA, DNA:RNA,DNA:DNA. For hybrids of greater than 100 nucleotides in length,equations for calculating T_(m) have been derived (see Sambrook et al.,supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e.,oligonucleotides, the position of mismatches becomes more important, andthe length of the oligonucleotide determines its specificity (seeSambrook et al., supra, 11.7-11.8). Preferably a minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; preferablyat least about 15 nucleotides; and more preferably the length is atleast about 20 nucleotides. In a specific embodiment, the term “standardhybridization conditions” refers to a T_(m) of 55° C., and utilizesconditions as set forth above. In a preferred embodiment, the T_(m) is60° C.; in a more preferred embodiment, the T_(m) is 65° C.

Integrin-binding peptides. Targeting peptide sequences include the wellknown integrin-binding peptides (see, e.g., U.S. Pat. No. 4,517,686;U.S. Pat. No. 4,589,881; U.S. Pat. No. 4,661,111; U.S. Pat. No.4,578,079; U.S. Pat. No. 4,614,517; U.S. Pat. No. 5,453,489; U.S. Pat.No. 5,627,263). Other useful targeting peptides are described inInternational Patent Publication WO 98/17242 and European PatentApplication EP 773 441.

Concerning the targeting of integrins, as noted above there are manypublications describing peptides that bind specific integrins, some ofwhich are overexpressed in different tumor or cell types. In a specificembodiment, αv integrins (and consequently actively dividing and/ormotile cells including tumor cells and its metastases, tumorvasculature, activated endothelial, activated smooth muscle cells,skeletal muscle cells, etc.) are targeted, e.g., through a peptideselected by phage display. Naturally, any peptide specific of integrins,e.g., as described in the literature, can be used in the invention. Thetargeting of integrins could also favor the internalization of thevirus, and could be a mean to restore the infectivity of Ad withshortened fibers.

Urokinase receptor targeted peptides. In a specific embodimentexemplified infra, peptides that target a receptor for urokinase-typeplasminogen activator (UPAR; e.g., CD87) were selected from variouspublications. Naturally, there is a much more exhaustive list ofpeptides binding a UPAR. Any peptide binding UPAR could be used.

Basic targeting peptides. Specific embodiments of the invention, withparticular targeting peptides, are described in detail in Examples 4-6,infra. For example, a targeting peptide comprising primarily basic aminoacid residues, e.g., lysine, arginine, and histidine, and morepreferably either lysine or arginine, or both (see, e.g., WO 97/20051)was used in the examples, infra. In another example, ligands binding toheparan sulfate proteoglycans such as the arginine-leucine repeatedmotif RRLLRRLLRR, described in the RPR patent application WO95/21931,and the peptide fragment KRGPRTHYGQK from the FGF-1 binding domain toheparin (Digabriele et al, 1998, Science, 393 :812-817) were used.

Also, sequences that bind to heparin or glycosaminoglycans may beinvolved in binding to a heparin-like receptor (Sawitzky et al., 1993,Med. Microbiol. Immunol., 182: 285-92). Similarly, so-called <<heparinbinding sequences>> may mediate the interaction of the peptide orprotein in which they are contained with other cell surface bindingsites, such as with cell surface heparan sulfate proteoglycan (Thompsonet al., 1994, J. Biol. Chem., 269: 2541-9).

Alternatively, the targeting amino acid sequence comprises two basicamino acids (frequently Arg) located about 20 Angstroms apart, facing inopposite directions of an alpha helix (Margalit et al., 1993, J. Biol.Chem., 268: 19228-31; Ma et al., 1994, J. Lipid Res., 35: 2049-2059).Other basic amino acids can be dispersed between these two residues,facing one side, while nonpolar residues face the other side, forming ahelical amphipathic structure with basic residues segregated to oneside.

Also, the targeting sequence can comprise common heparin binding motifspresent in fibronectin and heat shock proteins (Hansen et al., 1995,Biochim. Biophys. Acta, 1252: 135-45); insertions of 7 residues ofeither Lys or Arg, or mixtures of Lys and Arg (Fromm et al., 1995, Arch.Biochem. Biophys., 323: 279-87); the common basic C-terminal region ofIGFBP-3 and IGFBP-5 of about 18 amino acids and which comprises aheparin binding sequence (Booth et al., 1995, Growth Regul., 5: 1-17);either one or both of the two hyaluronan (HA) binding motifs locatedwithin a 35 amino acid region of the C-terminus of the HA receptor RHAMM(Yang et al., 1994, J. Cell Biochem., 56: 455-68); a synthetic peptide(Ala347-Arg361) modeled after the heparin-binding form of Staphylococcusaureus vitronectin comprising heparin-binding consensus sequences (Lianget al., 1994, J. Biochem., 116: 457-63); any one or more of five heparinbinding sites between amino acid 129 and 310 of bovine herpesvirus 1glycoprotein gIII or any one of four heparin binding sites between aminoacids 90 and 275 of pseudorabies virus glycoprotein gIII (Liang et al.,1993, Virol., 194: 233-43); amino acids 134 to 141 of pseudorabies virusglycoprotein gIII (Sawitzky et al., 1993, Med. Microbiol. Immunol., 182:285-92); heparin binding regions corresponding to charged residues atpositions 279-282 and 292-304 of human lipoprotein lipase (Ma et al.,supra); a synthetic 22 residue peptide, N22W, with a sequence modeledafter fibronectin and which exhibits heparin binding properties (Inghamet al., 1994, Arch. Biochem. Biophys., 314: 242-246); the motif presentin the ectodomain zinc binding site of the Alzheimer beta-amyloidprecursor protein (APP), as well as various other APP-like proteins,which modulates heparin affinity (Bush et al, 1994, J. Biol. Chem., 229:26618-21), 8 amino acid residue peptides derived from the cross-regionof the laminin A chain (Tashiro et al., 1994, Biochem. J., 302: 73-9);peptides based on the heparin binding regions of the serine proteaseinhibitor antithrombin III including peptides F123-G148 and K121-A134(Tyler-Cross et al., 1994, Protein Sci., 3: 620-7); a 14 K N-terminalfragment of APP and a region close to the N-terminus (i.e., residues96-110) proposed as heparin binding regions (Small et al., 1994, J.Neurosci., 14: 2117-27); a stretch of 21 amino acids of the heparinbinding epidermal growth factor-like growth factor (HB-EGF)characterized by a high content of lysine and arginine residues(Thompson et al., 1994, J. Biol. Chem, 269: 2541-9); a 17 amino acidregion comprising the heparin binding region of thrombospondin andcorresponding to a hep 1 synthetic peptide (Murphy-Ullrich et al., 1993,J. Biol. Chem., 268: 26784-9); a 23 amino acid sequence (Y565-A587) ofhuman von Willebrand factor that binds heparin (Tyler-Cross et al.,1993, Arch. Biochem. Biophys., 306: 528-33); the fibronectin-derivedpeptide PRARI, and larger peptides comprising this motif, that bindheparin (Woods et al., 1993, Mol. Biol. Cell., 4:605-613); the heparinbinding region of platelet factor 4 (Sato et al., Jpn. J. Cancer Res.,84: 485-8); and K18K sequence in the fibroblast growth factor receptortyrosine kinase transmembrane glycoprotein (Kan et al., 1993, Science259: 1918-21).

Identification of targeting peptide sequences. In another embodiment,targeting peptides can be derived from various types of combinatoriallibraries, using well known strategies for identifying ligands (see U.S.Pat. No. 5,622,699 and International Patent Application No.PCT/US96/14600). One approach uses recombinant bacteriophage to producelarge libraries. Using the “phage method” (Scott and Smith, 1990,Science 249:386-390; Cwirla, et al., 1990 Proc. Natl. Acad. Sci.,87:6378-6382; Devlin et al., 1990, Science, 249:404-406), very largelibraries can be constructed (10⁶-10⁸ chemical entities). A secondapproach uses primarily chemical methods, of which the Geysen method[Geysen et al., 1986, Molecular Immunology 23:709-715; Geysen et al.,1987, J. Immunologic Method 102:259-274) and the method of Fodor et al.(1991, Science 251:767-773) are examples. Furka et al. (1988, 14thInternational Congress of Biochemistry, Volume Ê5, Abstract FR:013;Furka, 1991, Int. J. Peptide Protein Res. 37:487-493), Houghton (U.S.Pat. No. 4,631,211, issued December 1986) and Rutter et al. (U.S. Pat.No. 5,010,175, issued Apr. 23, 1991) describe methods to produce amixture of peptides that can be tested as targeting sequences. Inanother aspect, synthetic libraries (Needels et al., 1993, Proc. Natl.Acad. Sci. USA 90:10700-4; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci.USA 90:10922-10926; Lam et al., International Patent Publication No. WO92/00252; Kocis et al., International Patent Publication No. WO9428028), and the like can be used to screen for targeting peptides.

Uses of the Vectors of the Invention

The references provided above regarding preparation of adenovirusvectors describe various uses for such vectors.

Targeted introduction of therapeutic genes into malignant cells in vivocan provide an effective treatment of tumors. Several treatmentmodalities have been attempted. For example, one treatment involves thedelivery of normal tumor suppressor genes (e.g., p53, retinoblastomaprotein, p16, etc.) and/or inhibitors of activated oncogenes into tumorcells. A second treatment involves the enhancement of immunogeneity oftumor cells in vivo by the introduction of cytokine genes. A thirdtreatment involves the introduction of genes that encode enzymes capableof conferring to the tumor cells sensitivity to chemotherapeutic agents.The herpes simplex virus-thymidine kinase (HSV-tk) gene can specificallyconvert a nucleoside analog (ganciclovir) into a toxic intermediate andcause death in dividing cells. It has recently been reported by Culveret al. (Science, 1992, 256:1550-1552) that after delivery of the HSV-tkgene by retroviral transduction, subsequent ganciclovir treatmenteffectively caused brain tumor regression in laboratory animals. U.S.Pat. No. 5,631,236 by Woo et al. describes gene therapy for solid tumorswith an adenovirus vector that encodes HSV-tk or VZV-tk.

In a preferred embodiment, a vector of the invention can be used totarget a nucleic acid of interest to the tumor itself, its metastases orthe tumor vasculature, e.g., by using peptides that bind to a UPAR. In apreferred embodiment, such vectors encode genes for inhibitors ofangiogenesis or an anti-angiogenic factor. An “anti-angiogenic factor”is a molecule that inhibits angiogenesis, particularly by blockingendothelial cell migration. Such factors include fragments of angiogenicproteins that are inhibitory (such as the amino-terminal fragment ofurokinase), angiogenesis inhibitory factors, such as angiostatin andendostatin; soluble receptors of angiogenic factors, such as theurokinase type receptor or FGF/VEGF receptor; molecules which blockendothelial cell growth factor receptors [O'Reilly et. al. Cell88:277-285 (1997); O'Reilly, Nat. Med. 2:689-692 (1996)], and Tie-1 orTie-2 inhibitors. Generally, an anti-angiogenic factor for use in theinvention is a protein or polypeptide, which may be encoded by a genetransfected into tumors using the vectors of the invention. For example,the vectors of the invention can be used to deliver a gene encoding ananti-angiogenic protein into a tumor, its metastases or the tumorvasculature in accordance with the invention. Examples ofanti-angiogenic factors include, but are not limited to, the aminoterminal fragment (ATF) of urokinase, containing the EGF-like domain(e.g., amino acid residues about 1 to about 135 of ATF); ATF provided asa fusion protein, e.g., with immunoglobulin or human serum albumin[WO93/15199]; angiostatin [O'Reilly et al., Cell 79:315-328 (1994)];tissue inhibitors of metalloproteinases [Johnson et al., J. Cell.Physiol. 160:194-202 (1994)]; or inhibitors of FGF or VEGF such assoluble forms of receptors for angiogenic factors, including but notlimited to soluble VGF/VEGF receptors, and soluble urokinase receptors[Wilhem et al., FEBS Letters 337:131-134 (1994)].

In another preferred embodiment, a vector of the invention can be usedto target migrating smooth muscle cells to inhibit post-angioplasticrestenosis. An example of the use of an adenovirus to inhibit restenosisby delivery of a suicide gene is disclosed in WO96/05321. Use of anadenovirus encoding a GAX protein (growth arrest protein) to inhibitvascular smooth muscle cell proliferation and restenosis is disclosed inWO96/30385. Other genes can be cytotoxic genes (HSV thymidine kinase),metalloproteinases inhibitors (TIMP), endothelial NOS or atheroscleroseprotecting factors (e.g. ApoE).

A vector of the invention in which a muscle or a brain specific peptidehas been included can also be used to selectively deliver protecting orregenerating growth factors for central nervous system (CNS) disorders.Examples of the use of adenoviruses to deliver genes to the CNS aredisclosed in WO94/08026, WO95/25804 and WO95/26408.

A vector of the invention in which a skeletal muscle- orcardiac-specific peptide has been included can also be used toselectively deliver angiogenic factors (e.g; members of the VEGF, FGF,angiopoietin famillies), cell survival-promoting factors (e.g. membersof the akt/PKB familly), genes involved in the energetic metabolism(e.g. phospholamban or adenylyl cyclase), cytokins and their receptors(e.g. IL-6, IL 10, CXCR4, CXCR1, sdf1, MCP 1, GM-CSF and genesprotecting against apoptose (akt) useful for the treatment of peripheralartery diseases or coronary artery diseases.

In a more general way the vectors of the present invention to deliver totargeting cells genes enzymes, blood derivatives, hormones such asinsulin or growth hormone, lymphokines: interleukins, interferons, TNF,and the like (French Patent No. 92 03120), growth factors, for exampleangiogenic factors such as VEGF or FGF, neurotransmitters or precursorsthereof or synthesis enzymes, trophic, in particular neurotrophic,factors for the treatment of neurodegenerative diseases, of traumaswhich have damaged the nervous system, or of retinal degeneration: BDNF,CNTF, NGF, IGF, GMF, IFGF, NT3, NT5, HARP/pleiotrophin, or bone growthfactors, haematopoietic factors, and the like, dystrophin or aminidystrophin (French Patent No. 91 11947), genes encoding factorsinvolved in coagulation: for example, factors VII, VIII and IX, suicidegenes (e.g. thymidine kinase and cytosine deaminase), genes forhaemoglobin or other protein carriers, genes corresponding to theproteins involved in the metabolism of lipids, of the apolipoproteintype chosen from apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III,D, E, F, G, H, J and apo(a), metabolic enzymes such as, for example,lipoprotein lipase, hepatic lipase, lecithin-cholesterolacyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidyl acidphosphatase, or lipid transfer proteins such as the cholesterol estertransfer protein and the phospholipid transfer protein, an HDL-bindingprotein or a receptor chosen, for example, from the LDL receptors, theremnant chylomicron receptors and the scavenger receptors, and the like.It is possible to add, in addition, leptin for the treatment of obesity.

Among the other proteins or peptides which may be encoded by the gene ofthe targeted vector, it is important to underline antibodies, thevariable fragments of single-chain antibody (ScFv) or any other antibodyfragment possessing recognition capacities for its use in immunotherapy,for example for the treatment of infectious diseases, of tumours, ofautoimmune diseases such as multiple sclerosis (which may involve theuse of antiidiotype antibodies). Other proteins of interest are, in anonlimiting manner, soluble receptors such as, for example, the solubleCD4 receptor or the soluble receptor for TNF which may be used forexample for anti-HIV therapy, the soluble receptor for acetylcholinewhich may be used for example for the treatment of myasthenia; substratepeptides or enzyme inhibitors, or peptides which are agonists orantagonists of receptors or of adhesion proteins such as, for example,for the treatment of asthma, thrombosis and restenosis; artificial,chimeric or truncated proteins. Among the hormones of interest, theremay be mentioned insulin in the case of diabetes, growth hormone andcalcitonin.

The said gene may also be replaced by an antisense sequence or genewhose expression in the target cell makes it possible to control theexpression of genes or the transcription of cellular mRNAs. Suchsequences may, for example, be transcribed in the target cell into RNAcomplementary to cellular mRNAs and thus block their translation intoprotein, according to the technique described in European Patent No. 140308. The therapeutic genes may also comprise the sequences encodingribozymes, which are capable of selectively destroying target RNAs(European Patent No. 321 201).

EXAMPLES

The present invention may be better understood by reference to thefollowing non-limiting Examples, which are provided as exemplary of theinvention. All the modifications presented in the examples can becombined when not cited.

Example 1 Manipulation of the Hexon HVR5 Loop of Ad5

The present example demonstrates that manipulation of the HVR5 loop ofAd5 hexon from amino acids (aa) 269 to 281, keeping intact the mostconserved residues at the extremities of the loop (a serine in position268, and a proline in position 282), unexpectedly provides for effectiveadenovirus tropism engineering. The sequence removed from the Ad5 hexonwas: TTEAAAGNGDNLT (SEQ ID NO: 25) (i.e., our constructs naturallydisplay a threonine to alanine substitution at hexon residue 273 ascompared to the published refence sequence of Ad5), and the flankingsequences conserved were FFS (upstream) and PKVV (downstream).

Materials and Methods

Construction of shuttle plasmids for the manipulation of the HVR5 loopof the hexon. A first intermediate plasmid IE28 containing the flankingregions of the HVR5 loop and in which the HVR5 loop was replaced withthe xylE marker gene from Pseudomones putida (Zukowski et al., 1983,PNAS 80:1101-1105) was made using the following two primer pairs:hex-19243G (5′ATGGGATGAAGCTGCTACTG- 3′) (SEQ ID NO:26) and hex-19623D(5′tcgcgaGAAAAATTGCATTTCCACTT-3′), (SEQ ID NO:27) and hex-19685G(5′CCTAAGGTGGTATTGTACAG-3′) (SEQ ID NO:28) and hex-20065D(5′AGCAGTAATTTGGAAGTTCA-3′). (SEQ ID NO:29)

These primers were used to amplify portions of the hexon genecorresponding respectively to nucleotides 19245 to 19639 and 19685 to20084 of the Ad5 genome (Genbank access number M73260) according tostandard PCR techniques. Primer hex-19623D contains the restriction siteNruI and the primer hex-19685G was slightly modified with respect to Ad5sequence to create a Bsu36I site without modifying the protein sequenceof the hexon (nucleotide 19690: A to G). Each PCR product was cloned inthe plasmid pCRII (Invitrogen) to generate the plasmids IE21 and IE22,respectively. Proper cloning was confirmed by DNA sequencing.

The plasmid pαxylEΩ (Frey et al., 1988, Gene 62:237-247) containing theexpression cassette of the xylE gene was restricted with EcoRI,blunt-ended with the T4 DNA polymerase, and digested with HindIII. ThisDNA fragment was cloned into the blunt-ended BamHI-HindIII IE21 plasmid,resulting in plasmid IE26. Finally, the HindIII-XbaI fragment from IE26and the XhoI-HindIII fragment from IE22 were cloned into the SalI-XbaIcleaved plasmid pXL2756 previously described (Crouzet et al., 1997, PNAS94: 1414-1419) to generate the shuttle plasmid IE28.

All the plasmids modified in the HVR5 loop were derived from plasmidIE28 by replacement of the xylE gene with double-strandedoligonucleotides. Briefly, complementary single-strandedoligonucleotides were annealed to form duplexes and cloned into theNruI-Bsu36I digested IE28, except for the IE31 plasmid, which wasobtained by ligation of the double-stranded oligonucleotide with theBsrGI-NruI cleaved IE28. Phenotypic screening based on the yellowstaining of bacteria expressing xylE after spraying with 0.5M catecholwas used (Zukowski et al., 1983 supra.). The following table indicatesthe list of the oligonucleotides used and the names of the correspondingshuttle plasmids: TABLE 1 Oligonucleotides used to produce specifichexon insert plasmid constructs. SEQ shuttle ID plasmid oligonucleotidesNO: IE30 (Ad2 HVR5) 5′- AATACTACCTCTTTGAACGACCGGCAAGGCAATGCTACTAAACC-3′30 5′- TTAGGTTTAGTAGCATTGCCTTGCCGGTCGTTCAAAGAGGTAGTATT-3′ 31 IE31(epitope 5′-AATCTAGACTCTTTGGAACAACCTACTACTCGCGCTCAAAAACCACGTCTAGATTT-3′32 from poliovirus5′-GTACAAATCTAGACGTGGTTTTTGAGCGCGAGTAGTAGGTTGTTCCAAAGAGTCTAGATT-3′ 33type 3)* IE32 5′- TCAACCACTATAAACATTCC-3′ 34 (Ad 30 HVRS 5′-TTAGGAATGTTTATAGTGGTTGA-3′ 35 IE335′-ACTCCTGGCGCAAATCCTCCAGCAGGCGGTAGTGGAAACGAAGAATACAAACC-3′ 36 (Ad 19HVR5) 5′-TTAGGTTTGTATTCTTCGTTTCCACTACCGCCTGCTAGGAGGATTTGCGCCAGGAGT-3′ 37IE35(epitope from 5′-GATAACCCAGCGTCGACCACGAATAAGGATAAGCTACC-3′ 38poliovirus type 1) 5′-TTAGGTAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATC-3′ 39(5)) IE37(epitope from 5′-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCC-3′ 40poliovirus type 1 5′-TTAGGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3′ 41IE40(epitope from 5′-TCTGATAACCCAGCGTCGACCACGAATAAGGAAAGCC-3′ 42poliovirus type 1) 5′-TTAGGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCAGA-3′ 43IE41 (epitope from 5′- GGATCTGATAACCCAGCGTCGACCACGAATAAGGATAAGCC-3′ 44poliovirus type 1) 5′-TTAGGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCAGATCC-3′ 45IE43(epitope from 5′-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTAGGTGGCCC-3′46 poliovirus type 1)5′-TTAGGGCCACCTAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3′ 47 IE44(epitopefrom 5′-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTAGGTTCTCC-3′ 48 poliovirustype 1) 5′-TTAGGAGAACCTAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3′ 49IE45(epitope from 5′-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTATCTCC-3′ 50poliovirus type 1) 5′-TTAGGAGATAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3′51 IE46(epitope from 5′-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTATCTCC-3′52 poliovirus type 1)5′-TTAGGACCAGATAGCTTATCCTTATTCGTGGTCGACGCTGGGTTATCTCC-3′ 53 IE47(epitopefrom 5′-GGAGATAACCCAGCGTCGACCACGAATAAGGATAAGCTATCTAGTCC-3′ 52 poliovirustype 1) 5′-TTAGGACTAGATAGCTTATCCTTATTCGTGGTCGACCGCTGGGTTATCTCC-3′ 55*Crompton, et al., 1994, J. Gen. Vir. 75:133-139.

Construction of the associated plasmid backbones and viruses. Anintermediate plasmid backbone containing the xylE gene instead of theHVR5 loop of the hexon gene was constructed to facilitate subsequentmanipulation of the HVR5 loop. For this purpose, the shuttle plasmidIE28 was recombined with the plasmid backbone pXL3006 (this plasmidcontains a PacI-excisable E1E3-deleted adenoviral genome with a CMV-lacZexpression cassette in place of the E1 region) in the G4977 bacterialstrain according to the method described by Crouzet et al. (1997 supra.)to obtain the plasmid backbone IE28c, which differs from plasmidbackbone pXL3006 by the xylE-containing HVR5 sequence.

All the shuttle plasmids IE30 to IE47 were then recombined with theplasmid backbone IE28c using the “xylE screening” to get the plasmidbackbones IE30c to IE47c. After cleavage with the PacI enzyme, 2 μg (or5 μg) of these digested backbones were transfected in the 911 cells (or293 cells) using Lipofectamine (Gibco BRL) to generate the correspondingviruses AdIE30 to AdIE47. These HVR5-modified E1E3-deleted adenovirusestherefore express the same CMV-lacZ expression cassette.

All other HVR5-modified adenoviruses (e.g., displaying uPAR- orintegrin-binding peptides; see examples thereafter) were constructed bythe same strategy.

Cells and antibodies. 293 and W162 cells were maintained in MEM (GibcoBRL) supplemented with 10% fetal calf serum. 911 cells (Fallaux et al.,1996, Hum. Gene Ther. 7:215) were grown in DMEM supplemented with 10%fetal calf serum. The C3 monoclonal antibody (C3 mAb) directed againstthe poliovirus type 1 described in Blondel et al, 1983, Virology 126:707was provided by Dr. R. Crainic (Pasteur Institute, Paris, France). L5rabbit polyclonal antibodies directed against the whole Ad5 capsid wereproduced in RPR-Gencell's facilities (RPR SA, Vitry, France).

Viruses. All the viruses were amplified in E1-transcomplementing cells(e.g., 293 cells) according to classical methods. The pattern/identityof the viruses was controlled by restriction analysis and sequencing ofthe inserts on viral DNA obtained using the Hirt procedure. Viral stockswere prepared in 293 cells, purified by CsCl gradient, desalted usingPD10 columns (Pharmacia) and stored in PBS supplemented with 10%glycerol at −80° C. Biological quantification was carried out bynumbering the plaque-forming units (PFU) on 911 cells and/or numberingthe lacZ-transducing units (TDU) two days post-infection of W162 cellsfollowing X-Gal staining (Dedieu et al. 1997, J. Virol. 71 :4626).Physical quantification was carried out by anion-exchange by numberingthe viral particles (VP).

Neutralization test. One-half μl of anti poliovirus C3 monoclonalantibody were incubated in PBS for 1 h at 37° C. with 10⁵ TDU ofpurified virus, and the mix was then absorbed onto W162 cells in a 6wells-plate for a further 1 h at 37° C. Cells were then washed twicewith PBS, and fresh medium was added to the cells which were incubatedat 37° C. for 2 days. Cell monolayers were fixed with formaldehyde(0.37%)-glutaraldehyde (0.2%), X-Gal stained, and blue cells werecounted.

Immunoprecipitation protocol Viral particles (10¹⁰) of CsCl-purifiedvirus were resuspended in 400 μl of non-denaturing incubation buffer (50mM Tris pH7.5, 150 mM NaCl, 0.05% NP40) and then incubated with 0.1 μlof anti poliovirus C3 monoclonal antibody for 1 h at 4° C. Four hundredμl of protein A-Sepharose previously equilibrated with incubation bufferwas then added, and further incubated for 1 h at 4° C. Followingincubation, the mix was spun down by brief centrifugation in amicrocentrifuge. The pellet was washed twice with 1 ml of incubationbuffer and once with 1 ml of 10 mM Tris pH7.5, 0.1% NP40 for 20 min at4° C. The pellet containing the protein A-antibody-virus complex wasresuspended in 50 μl of Laemmli buffer 1×, boiled 2 min and thesupernatant was collected after a brief centrifugation. Ten μl ofsupernatant was analyzed by SDS-polyacrylamide gel electrophoresis(Novex). Western blot was carried out using the L5 rabbit polyclonalserum directed against the whole Ad5 capsid according to the ECLprocedure (Amersham).

Results

Construction strategy for modification of the HVR5 loop. Two series ofconstructions were made. The first one was designed to assess the“capacity” of the Ad5 HVR5 loop for foreign sequences, by replacement ofthis loop by the HVR5 loops from other Ad serotypes, which greatlydiffer in size. TABLE 2 Ad5-based adenoviruses with heterospecific HVR5loops. HVR5 sequence replacing the SEQ Virus sequence of HVR5 loop ofAd5 ID NO: Ad IE 30 Ad2:14 aa NTTSLNDRQGNATK 56 Ad IE 32 Ad30:6 aaSTTINI 57 Ad IE 33 Ad 19:17 aa TPGANPPAGGSGNEEYK 58

As a viability control, we introduced the modification published byCrompton et al. (1994, J. Gen. Virol. 75:133-139) in which a poliovirustype 3 epitope was introduced in place of a larger deletion encompassingHVR5. Importantly, the crompton virus was initially constructed byhomologous recombination between a plasmid containing the Ad2 modifiedhexon and an Ad5-based adenoviral genome. This virus therefore displaysa chimeric hexon protein between Ad2 and Ad5 in which a 4-residue largerHVR5 deletion has been substituted for a foreign peptide. To reproducethe Crompton et al. construct as closely as possible, an Ad5-based virus(Ad IE31) was constructed by the EDRAG technology in which the foreignpeptide of Crompton et al. was introduced in place of hexon residues 269to 285 (TTEAAAGNGDNLTPKVV; SEQ ID NO:59) of Ad5 instead of TTEAAAGNGDNLT(SEQ ID NO:60). TABLE 3 Insert of control virus AdIE31. Virus sequencereplacing the HVR5 loop of AdS Ad IE31 NLDSLEQPTTRAQKPRLD (SEQ ID NO:61)

In the second series of constructs, the HVR5 loop (aa 269 to 281) wasreplaced by a neutralizing linear epitope of poliovirus type 1(DNPASTTNKDK; SEQ ID NO. 62). This model binding-peptide was inserted invarious neighboring contexts (i.e., various linkers composed of leucineand/or glycine and/or serine residues were used) to assess theirimportance for virus viability and peptide accessibility. TABLE 4Insertions of a poliovirus type 1 epitope in the HVR5 loop. upstreamdownstream SEQ Virus linker epitope used linker ID NO: Ad IE35 noneDNPASTTNKDK L 63 Ad IE37 G DNPASTTNKDK none 64 Ad IE40 S DNPASTTNKDKnone 65 Ad IE41 G S DNPASTTNKDK none 66 Ad IE43 G DNPASTTNKDK LG G 67 AdIE44 G DNPASTTNKDK LG S 68 Ad IE45 G DNPASTTNKDK LS 69 Ad IE46 GDNPASTTNKDK LSG 70 Ad IE47 G DNPASTTNKDK LS S 71

Viability of the viruses. The three viruses with Ad2, Ad19, or Ad30 HVR5loops instead of the Ad5 HVR5 loop, and the nine chimeric viruscontaining the poliovirus type 1 epitope were viable. No loss ofproductivity was observed. Unexpectidely, the control virus Ad IE31could not be recovered despite intensive efforts.

Assessment of poliovirus epitope accessibility by immunoprecipitationassay. Immunoprecipitation experiments were performed on the virusescarrying the poliovirus epitope using a cognate antipoliovirusmonoclonal antibody (C3 mAb) in non-denaturing conditions. The followingtable 5 summarizes the data obtained: TABLE 5 Immunoprecipitation withC3 mAb. Virus peptide inserted in the HVR5 loop Immunoprecipitation AdIE35 DNPASTTNKDK-L − Ad IE37 G-DNPASTTNKDK − Ad IE40 S-DNPASTTNKDK − AdIE41 GS-DNPASTTNKDK − Ad IE43 G-DNPASTTNKDK-LGG ++ Ad IE44G-DNPASTTNKDK-LGS ++ Ad IE45 G-DNPASTTNKDK-LS ++ Ad IE46G-DNPASTTNKDK-LSG ++ Ad IE47 G-DNPASTTNKDK-LSS ++

The presence of a linker downstream of the poliovirus epitope was thusfound critical for C3 mAb binding to the modified viral capsids, mostlikely because it allows proper presentation and/or accessibility of thebinding peptide at the hexon surface. When the modified viruses weredenatured prior to immunoprecipitation, all of them were efficientlyimmunoprecipitated by C3 mAb (not shown).

Assessment of poliovirus epitope functionality by neutralization assay.As the C3 mAb is neutralizing for poliovirus infection, we anticipatedthat it could also neutralize the infectivity of Ad carrying thepoliovirus epitope. Incubation of C3 mAb with the whole series of Ad IEviruses was performed in PBS (i.e., under native conditions) prior toinfection of W162 monkey cells. The data are shown in FIG. 1. These datacorrelate perfectly with the immunoprecipitation results, i.e., all theviruses that could bind C3 mAb in non-denaturating conditions were alsoneutralized by this antibody.

Discussion

The data show that modification of the HVR5 loop of adenovirus can allowfunctional and specific interaction of the modified hexons with aspecific binding protein. Particular modes of insertion are howeverrequired as the data showed that accessibility (immunoprecipitationassay) and functionality (neutralization assay) of the binding peptideepitope were dependent on minimal spacer/neighboring sequences.

Contrasting with the conclusion of Crompton et al., our results alsoshow that the deletion of aa 269 to 285 of Ad5 hexon is deleterious forvirus growth and/or viability. This can be explained by the fact thatthe residues 282 to 285 are localized in the bêta-strand locateddownstream of the HVR5 loop, which is probably essential for thestructure of the hexon as a monomer or a trimer. Therefore, the presentapproach, which is more precise and rigorous than that described inCrompton et al., unexpectedly overcame the disadvantages of thatreference and provided viable viruses equipped with a modified tropism(see below).

Example 2 Manipulation of the Fiber HI Loop

Deletion of the HI loop of the Ad5 fiber was performed: the sequenceremoved from the fiber knob includes residues 538 to 548 (i.e., sequenceGTQETGDTTPS) (SEQ ID NO:72). Its flanking sequences are thus TLN(upstream) and AYS (downstream).

Materials and Methods

Construction of shuttle plasmids for the manipulation of the HI loop ofthe fiber. A first intermediate plasmid pJD3 containing the flankingregions of the HI loop and in which the HI loop was replaced with thexylE gene was made using the following two primer pairs: (SEQ ID NO:73 )HIgul (5′-CAGCTCCATCTCCTAACTGTAGACTAAATG-3′) and (SEQ ID NO:74 ) HIgul(5′-GGTTACCGGTTTAGTTTTGTCTCCGTTTAA-3′) and (SEQ ID NO:75 ) HIdul(5′-AGCGCTTACTCTATGTCATTTTCATGGGAC-3′) and (SEQ ID NO:76 ) HIddl(5′-GAGTTTATTAATATCACTGATGAGCGTTTG-3′).

These primer pairs were used to amplify portions of the fiber and E4orf7genes corresponding respectively to nucleotides 32255 to 32634 and 32712to 33090 of the Ad5 genome (Genbank access number M73260) according tostandard PCR techniques. The primers HIgd1 and HIdu1 are designed insuch a way that they create the restriction sites BstEII and Eco47III,respectively, without modifying the fiber protein sequence at theimmediate vicinity of the HI loop. These sites were further used fordirect cloning of foreign peptides into HI (see below and table 5).

Each PCR product was cloned into the plasmid PCR2.1 (Invitrogen) togenerate the plasmids PCR2.1-H4 and PCR2.1-I2, respectively, andsequenced.

The plasmid pαxylEΩ containing the expression cassette for the xylE genewas restricted with EcoRI, blunt-ended with the T4 DNA polymerase, anddigested with HindIII. This DNA fragment was subcloned intoHindIII-restricted PCRII (Invitrogen) resulting in the plasmid IE23. ANsiI-XhoI fragment of IE23 was introduced into the NsiI-XhoI digestedPCR2.1-H4 plasmid, resulting in the plasmid pJD2. Finally, the SacI-XbaIfragment from pJD2 and the SpeI-XhoI fragment from PCR2.1-I2 were clonedinto the previously described (Crouzet et al., 1997, supra.) plasmidpXL2756 cleaved by SacI-SalI to generate the shuttle plasmid pJD3.

All the plasmids modified in the HI loop were derived from plasmid pJD3by replacement of the xylE gene with double-stranded oligonucleotides.Briefly, complementary single-stranded oligonucleotides were annealed toform duplexes and cloned into BstEII-Eco47III digested pJD3. Aphenotypic screening based on the yellow staining of bacteria expressingxylE after spraying with 0.5M catechol was used. The following tableindicates the list of the oligonucleotides used and the name of thecorresponding shuttle plasmids: TABLE 5 Examples of oligonucleotidesused to replace the HI loop. SEQ Shuttle ID plasmid oligonucleotides NO:pJD7 (HI 5′-GTAACACTAACCATTACACTAAACGGTACCCAGGAAACAGGAGACACAACTCCAAGT-3′ 77 loopfrom 5′- ACTTGGAGTTGTGTCTCCTGTTTCCTGGGTACCGTTTAGTGTAATGGTTAGT-3′ 78 Ad5)pJDS(HI 5′-GTAACACAACCATTACACTAAACGGTACCAGTGAATCCACAGAAACTAGCGAGGTAAGC-3′ 79 loopfrom 5′- GCTTACCTCGCTAGTTTCTGTGGATTCACTGGTACCGTTTAGTGTAATGGTTAGT-3′ 80Ad2) pJD6(HI 5′- GTAACACTAACCATTACACTAAACCAAGAAACACAATGTGAA-3′ 81 loopfrom 5′- TTCACATTGTGTTTCTTGGTTTAGTGTAATGGTTAGT-3′ 82 Ad9) pCF15′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGAGC-3′ 83(epitope5′-GCTCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTCTAATGGTTAGG-3′ 84 frompoliovirus type 1) pCF2 5′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGGGAAGC-3′ 85(epitope 5′-GCTTCCCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′3 86from poliovirus type I) pCF35′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGTCAAGC-3′87 (epitope5′-GCTTGACTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′ 88from poliovirus type 1 pCF45′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGGGCGGAAGC-3′89 (epitope5′-GCTTCCGCCCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′90 from poliovirus type 1) pCF55′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGTCATCTAGC-3′91 (epitope5′-GCTAGATGACTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′92 from poliovirus type 1) pCF65′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGGGATCCAGC-3′93 (epitope5′-GCTGGATCCCTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′94 from poliovirus type 1) pCF75′-GTAACCCTAACCATTACACTAAACGGTGATAACCCAGCGTCGACCACGAATAAGGATAAGTCAGGAAGC-3′95 (epitope5′-GCTTCCTGACTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′96 from poliovirus type I) pCF85′-GTAACCCTAACCATTACACAAACGGTGATAACCCAGCGTCGACCACCACGAATAAGGATAAG-3′ 97(epitope 5′-CTTATCCTTATTCGTGGTCGACGCTGGGTTATCACCGTTTAGTGTAATGGTTAGG-3′98 from poliovirus type I)

Construction of the associated plasmid backbones and viruses. Anintermediate plasmid backbone containing the xylE gene instead of the HIloop of the fiber gene was first constructed to facilitate subsequentscreening of any plasmid backbone displaying a modified HI loop. Forthis purpose, the shuttle plasmid pJD3 was recombined with the plasmidbackbone pXL3006 in the G4977 bacterial strain according to the methoddescribed by Crouzet et al., supra, to obtain the plasmid backbone pBX,which therefore displays a PacI-excisable E1 E3-deleted adenoviralgenome containing a CMV-lacZ expression cassette in place of E1, as wellas the xylE marker in the HI loop.

Shuttle plasmids pJD5, 7 and 6, and pCF1 to pCF8 were then recombinedwith the plasmid backbone pBX using the “xylE screening” to get theplasmid backbones pBV2, 5 and 9, and pBC1 to pBC8, respectively. Aftercleavage with the PacI enzyme, 2 μg (or 5 μg) of DNA were transfected inthe 911 cells (or 293 cells) using Lipofectamine (Gibco BRL) to generatethe corresponding viruses vBV2, 5 and 9, and vBC1 to vBC8.

Other methods. The cells, antibodies, viruses, immunoprecipitationassays, and neutralization assays were as described in Example 1, supra.

Results

Construction strategy for the HI loop insertion vectors. Two series ofconstructions were made. The first one was designed to assess the“capacity” of the Ad5 HI loop for foreign sequences, by replacement ofthis loop by the HI loops of other adenovirus serotypes, with somevariations in size. TABLE 6 Capacity insertions in the fiber protein HIloop. HI sequence replacing SEQ Virus sequence of the HI loop of Ad5 IDNO: VBV2 Ad2: 12 aa GTSESTETSEVS  99 VBV5 Ad5: 11 aa GTQETGDTTPS 100VBV9 Ad9: 6 aa QETQCE 101

In the second series of constructs, the HI loop was replaced by aneutralizing epitope of poliovirus type 1 (DNPASTTNKDK) (SEQ ID NO:62).Due to the very close 3D structures at the N-terminal sides of the Ad5HI loop and the poliovirus sequences in their nativeenvironment/protein, a minimal one residue linker (glycine) was addedusptream of the epitope. This was not the case downstream of theinsertion site for which spacers of different length and compositionwere included to assess their impact on virus growth and peptideaccessibility. TABLE 7 Insertions of a poliovirus type 1 epitope in thefiber protein HI loop. downstream Virus epitope used linker SEQ ID NO:vBC1 DNPASTTNKDK S 102 vBC2 DNPASTTNKDK G S 103 vBC3 DNPASTTNKDK S S 104vBC4 DNPASTTNKDK G G S 105 vBC5 DNPASTTNKDK S S S 106 vBC6 DNPASTTNKDK GS S 107 vBC7 DNPASTTNKDK S G S 108 vBC8 DNPASTTNKDK none 109

Viability of the viruses. The three control viruses with Ad2, Ad5 or Ad9HI loops instead of the Ad5 HI loop and the 8 chimaeric virus containingthe poliovirus epitope were viable. No loss of productivity wasobserved.

Assessment of poliovirus epitope accessibility by immunoprecipitationassay. Immunoprecipitation experiments of the viruses carrying thepoliovirus epitope were performed with the cognate antipoliovirus C3 mAbin non-denaturing conditions. The following table summarizes the data:TABLE 8 Recognition of the targeting sequence as detected byimmunoprecipitation with C3 mAb. Virus Peptide inserted in the HI loopImmunoprecipitation VBC1 G-DNPASTTNKDK-S − VBC2 G-DNPASTTNKDK-GS +/−VBC3 G-DNPASTTNKDK-SS + VBC4 G-DNPASTTNKDK-GGS ++ VBC5 G-DNPASTTNKDK-SSS++ VBC6 G-DNPASTTNKDK-GSS ++ VBC7 G-DNPASTTNKDK-SGS ++ VBC8G-DNPASTTNKDK −

These data show that, as for HVR5 loop insertion, binding of the C3 mAbantibody to the modified capsids was criticaly dependent on the presenceof a spacer sequences of minimal length. In particular, a downstreamlinker of 3 residues conferred the most efficient binding. When theviruses were denatured prior to the immunoprecipitation assay, they wereall able to bind the C3 mAb antibody (not shown).

Assessment of poliovirus epitope functionality by neutralisation assay.The ability of C3 mAb to neutralize infectivity of the fiber-modifiedviruses was then assessed to determine if it also correlated with theimmunoprecipitation data as exemplified in Example 1 for thehexon-modified capsids. However, unlike the results with hexonmodification, there was no inhibition of infectivity followingincubation of any of the fiber-modified virus with C3 mAb (data notshown). Different explanations can be suggested. In particular, thereare only 12 fiber trimers on the virion surface versus 240 hexontrimers. Therefore, the density of antibody binding to thehexon-modified capsids may have decreased virus infectivity eitherdirectly, or following steric hindrance of the RGD motif of the pentonbase so integrin-mediated internalisation was affected. In any case,C3mAb specific binding to the HI-modified capsids did not prevent nativeinteraction between the virus and its target cells.

Discussion

The data demonstrate that the HI loop of adenovirus can be replaced by aforeign peptide and that the corresponding viruses are: i) viable, andii) with no drastic effect on virus productivity. The data alsodemonstrate that specific recognition of the introduced peptide requiressuitable neighboring linkers (i.e., flexible linkers of minimal size).

Example 3 Modification of Fiber Protein Length—Short Fibers

Taking into account the comparison of the cell-binding pathway of Ad5 (along-fiber serotype) with other serotypes such as Ad9 (a short-fiberserotype), the length of the shaft of the fiber was modified bydesigning either an intertypic fiber (substitution of the Ad5 shaft bythe Ad3, another short-fiber serotype, shaft), or a shortened Ad5 shaftthat retained only 6 or 9 repeats instead of 22 in the native protein.

Materials and Methods

Construction of shuttle plasmids with shortened fibers. Three shuttleplasmids containing shortened fibers were constructed. Two of them (pSF1and pSF2) display major deletions within the fiber shaft, whereas thethird one (pIF1) harbors the Ad3 shaft instead of that of Ad5. Allconstructs therefore retain the Ad5 tail and knob domains.

To construct pSF1, two pairs of primers were used: (SEQ ID NO:110 ) 5M3g(5′-ATTTCTGTCGACTTTATTCAGCAGCACCTC-3′) and (SEQ ID NO:111 ) 5M3d(5′-GTTTGACTTGGTTTTTTTGAGAGGTGGGCT-3′), and (SEQ ID NO:112 ) 5M17g(5′-TTGGATATTAACTACAACAAAGGCCTTTAC-3′) and (SEQ ID NO:113 ). 5M17d(5′-GAAACTGGAGCTCGTATTTGACTGCCACAT-3′).

These primers were used to amplify portions of the fiber genecorresponding respectively to nucleotides 30885 to 31329 and 31936 to32351 of the Ad5 genome (Genbank access number M73260) according tostandard PCR techniques. The primers 5M3g and 5M17d slightly differ fromthe Ad5 sequence by respectively containing the restriction sites SalIand SacI. After digestion by SalI or SacI, these PCR products wereligated and cloned into the SacI-SalI cleaved pXL2756, resulting in thepSF1 plasmid which displays an in frame deletion encompassing shaftrepeats 4 to 16.

To construct pSF2, two pairs of primers were used: (SEQ ID NO: 114 )5M3g (5′-ATTTCTGTCGACTTTATTCAGCAGCACCTC-3′) and (SEQ ID NO: 115 ) 5M3d(5′-GTTTGACTTGGTTTTTTTGAGAGGTGGGCT-3′), and (SEQ ID NO: 116 ) 5M20g(5′-CTCAAAACAAAAATTGGCCATGGCCTAGAA-3′) and (SEQ ID NO: 117) 5M20d(5′-ATCCAAGAGCTCTTGTATAGGCTGTGCCTT-3′).

These primers were used to amplify portions of the fiber genecorresponding respectively to nucleotides 30885 to 31329 and 32110 to32530 of the Ad5 genome according to standard PCR techniques. Theprimers 5M3g and 5M20d slightly differ from the Ad5 sequence byrespectively containing the restriction sites SalI and SacI. Afterdigestion by SalI or SacI, these PCR products were ligated and clonedinto the SacI-SalI cleaved pXL2756, resulting in the pSF2 plasmid whichdisplays an in frame deletion encompassing shaft repeats 4 to 19.

The pIF1 plasmid was made using the gene SOEing method described byHorton et al (4) consisting of recombining DNA sequences by PCR withoutrelying on restriction sites. Five successive steps were necessary toconstruct the pIF1 plasmid, which contains an intertypic fiber gene,composed of the Ad5 tail and knob and the Ad3 shaft. A first PCR productcontaining the Ad5 fiber tail was amplified from the Ad5 genome usingthe primers: (SEQ ID NO:118 ) SOE35Tg(5′-TACAAGTCGACAACCAAGCGTCAGAAATTG-3′) and (SEQ ID NO:119 ) SOE35Td(5′-AAGACTTAAAACCCCAGGGGGACTCTCTTG-3′)

The primer SOE35Tg nearly matches the Ad5 nucleotides 30660 to 30689with a slight modification resulting in the creation of a SalI site. The15 underlined bases in the SOE35Td primer match with the sequence of thefirst repeat of the Ad3 fiber shaft and the 15 remaining basescorrespond to the sequence of the fiber tail end of Ad5.

A second PCR product containing the Ad3 fiber shaft was amplified fromthe Ad3 fiber gene using the primers: (SEQ ID NO: 120 ) SOE35Sg(5′-GAGAGTCCCCCTGGGGTTTTAAGTCTTAAA-3′) and (SEQ ID NO: 121 ) SOE35Sd(5′-GGTCCACAAAGTGTTATTTTTCAGTGCAAT-3′).

The underlined bases in both primers are contained in the Ad5 fiber gene(respectively within the tail and knob subdomains), the remaining onesare from the Ad3 shaft.

A third PCR product containing part of the Ad5 fiber knob was amplifiedfrom the Ad5 fiber gene using the primers (SEQ ID NO: 122 ) SOE35Kg(5′-CTGAAAAATAACACTTTGTGGACCACACCA-3′) and (SEQ ID NO: 123 ) SOE35Kd(5′-TCCTGAGCTCCGTTTAGTGTAATGGTTAGT-3′).

The underlined bases in the SOE35Kg primer fit with the sequence of thelast repeat of the Ad3 fiber shaft, whereas the remaining onescorrespond to the first nucleotides of the Ad5 fiber knob. The primerSOE35Kd nearly matches the Ad5 nucleotides 32634 to 32663 with a slightmodification resulting in the creation of a SacI site. The two first PCRproducts were mixed, denatured and reannealed under PCR conditions, sothat the top strand of the first product and the bottom strand of thesecond one overlap and act as primers on one another. The hybrid productwas formed when this overlap was extended by polymerase. Inclusion ofthe primers SOE35Tg and SOE35Sd in the SOE reaction caused therecombinant product to be PCR amplified right after it is formed (seefigure of Horton et al, previously cited). This resulted in a PCRproduct containing the Ad5 tail followed by the Ad3 shaft. Finally, thesame SOE procedure was carried out with this last PCR product and thethird PCR product using the primers SOE35Tg and SOE35Kd, giving rise toa DNA fragment containing an intertypic fiber composed of the Ad5 tail,the Ad3 shaft and part of the Ad5 knob, and flanked with the uniquerestriction sites SalI and SacI (FIG. 2). After digestion with theseenzymes, the final PCR product was cloned into the SalI-SacI cleavedpXL2756 plasmid, resulting in the pIF1 shuttle plasmid.

Plasmids pSF1, pSF2 and pIF1 were sequenced.

Construction of the associated plasmid backbones and viruses. The threeshuttle plasmids pSF1, pSF2 and pIF1 were recombined with the plasmidbackbone pXL3006 (contains a PacI-excisable E1 E3-deleted recombinantadenoviral genome with a CMV-lacZ expression cassette in place of the E1region) in the G4977 bacterial strain according to the method describedby Crouzet et al., supra, to obtain the plasmid backbones pBS1, pBS2 andpBI1, respectively. After cleavage with PacI, 2 μg (or 5 μg) of DNA weretransfected in the 911 cells (or 293 or PER.6 cells) using Lipofectamine(Gibco BRL) to generate the corresponding viruses vBS1, vBS2 and vBI1.

The intermediate plasmid backbones AE31, AE32 and AE33 were alsoconstructed by homologous recombination between shuttle plasmid IE28 andthe pBI1, pBS1 and pBS2 plasmid backbones. These plasmid backbones whichcontain a xylE expression cassette instead of the HVR5 hexon looptogether with short-shafted fibers were further used to facilitate therecovery of plasmid backbones combining short-shafted fibers andHVR5-modified hexons (e.g., with insertion of UPAR- or integrin-bindingpeptides; see example 10).

In parallel, the intermediate plasmid backbones AE34, AE35 and AE36 wereconstructed by recombination between shuttle plasmid pJD3 and the pBI1,pBS1 and pBS2 plasmid backbones. These plasmid backbones were furtherused to facilitate the recovery of plasmid backbones combiningshort-shafted fibers and HI-modified fibers (e.g., with insertion ofUPAR- or integrin-binding peptides).

Other methods. The cells, antibodies, viruses, immunoprecipitationassays, and neutralization assays were as described in Example 1, supra.

Results and Discussion

Viruses with shortened fiber proteins are expected to increaseaccessibility of binding peptides exposed at the hexon surface and/orreducing wild-type (i.e., native) virus/cell interactions. Theseconstructs are summarized in Table 9. TABLE 9 Shortened fiber proteinadenoviruses. Virus structure of the chimeric fiber vBI1 Ad5 tail - Ad3shaft - Ad5 knob vBS1 Short-shafted fiber from Ad5 (deletionencompassing shaft repeats 4-16) vBS2 Short-shafted fiber from Ad5(deletion encompassing shaft repeats 4-19)

Viral productivity was drastically reduced in all three cases (althoughto different degrees), most likely because of the inability of themodified fibers to interact efficiently with its cellular receptor. Thatthe defect occurred at such an early stage is indeed supported by normalviral DNA replication parameters in 293-infected cells (i.e.,Xgal-positive), together with normal accumulation of viral lateproteins. Also, Western blotting under non-denaturing conditionsdemonstrated that proper trimerization of the modified fibers occurredin all three cases.

Although vBS1 binds less efficiently to CAR (Coxsackie and adenovirusesreceptor)-positive cells, it could however be amplified in PER.C6 up toa lab-scale stock. As shown in FIG. 3, it was also observed thatinfection of 293 cells with vBS1 resulted in a 10-fold decrease in celltransduction as compared to that of a control virus displaying thenative Ad5 fiber, which indicates a significant loss in Ad receptorbinding. In this experiment, 293 cells were incubated with PBS orpurified fiber knob (100 μg/ml) for 30 min at RT before infection at anmoi of 50 (VP/cell) for 30 min at RT. Cells were then washed twice withPBS and furter incubated 24 h at 37° C. in medium before preparation ofprotein extracts. Specific lacZ expression was then quantified (unitsper protein extract).

Based on these data, C57/B16 mice were injected iv with the vBS1construct or its control virus (unmodified shaft) to determine theprofile of lacZ expression in major organs. Liver, heart and lung wereanalyzed for β-galactosidase expression by histochemistry. Results arepresented in the following table 10 (% of XGal-positive cells mean andrange of values). TABLE 10 Profile of transgene expression in majororgans injected dose (VP) control virus vBS1 liver  3.10⁹ (n = 5) 15(10-20) 0 (0-0)   10¹⁰ (n = 5) 50 (50-60) 5 (2-5) 3.10¹⁰ (n = 5) 50(20-80)  15 (10-20) heart  3.10⁹ (n = 5) 0 0   10¹⁰ (n = 5) 0 0 3.10¹⁰(n = 5) 0 0 lung  3.10⁹ (n = 5) 0 0   10¹⁰ (n = 5) 0 0 3.10¹⁰ (n = 5) 00

These data indicate a dose-response effect for both adenoviruses in theliver, whereas no XGal-positive cells could be evidenced in the heartand lung tissues from either treated groups. Most interestingly,shortening of the fiber shaft resulted in a 10-fold decrease in livertransduction emphasizing the usefulness of manipulating the fiber shaftto direct infection mostly to the desired organs/cells in vivo, providedthe recombinant adenovirus has been equipped with an additional,CAR-independent, entry pathway.

Example 4 Specific Targeting of Cells Expressing a Urokinase-TypePlasminogen Activator Receptor

Various high affinity uPAR-binding peptides were included within thehexon HVR5 and the fiber HI loops, or added to the C-terminal end of thefiber protein. These peptides originate either from wild-type ATF(Rettenberg et al., 1995, Biol. Chem. Hoppe-Seyler 376:587-594) andmutant ATF (Magdolen et al., 1996, Eur. J. Biochem. 237:743-751), aphage library (Goodson et al., 1994, Proc. Natl. Acad. Sci.91:7129-7133), and an associated mutant, or human vitronectin (Waltz etal., 1997, J. Clin. Invest. 100:58-67). All viruses contain a geneexpression cassette (lacZ or Gax) inserted in place of the E1 genes.

The methods for insertion of these peptides in the hexon HVR5 loop andthe fiber protein HI loop were as described for the poliovirus epitopesin Examples 1 and 2, above. Shortening of the fiber protein was achievedas described in Example 3. Further Material and Methods are describedhereunder.

Cell Culturing of PERC6 Cells

PER.C6 cells were grown in Dulbecco's modified medium (DMEM), 10% FCSand 10 mM MgCl₂ in a 10% CO₂ atmosphere at 37° C. (Fallaux et al, 1998,Hum Gene Ther, 9 :1909-1917).

Recombinant Adenoviral Genomes

Recombinant adenoviral genomes were cloned into an RK2-derived plasmidby homologous recombinations in E. coli utilizing the EDRAG technologyas described in the French application FR 2 730 504. The technology wassimplified by replacing the ColE1 origin of replication by the origin ofR6K in the suicide shuttle, as described in WO 97/10343, which allowsrecombination in any recA⁺ E. coli strain. Suicide plasmids (alsoreferred to as shuttle plasmids) were constructed by inserted Ad5sequence at appropriate restriction sites and by modifying sequences bysequential PCR. The integrity of the EDRAG constructs was assessed byrestriction enzyme mapping and Southern analysis. The regions involvedin PCR amplification or homologous recombination were verified bysequence analysis.

Plasmid backbone pXL3215 is a 57.8 kb long RK2 derivative that containsa PacI-excisable E1 and E3-deleted Ad5-based genome (French applicationFR 2 730 504) with an E. coli lacZ gene under control of the RousSarcoma Virus promoter instead of the E1 region. Plasmid pXL3527 derivesfrom pXL3215 by exchanging the E. coli lacZ expression cassette by thehuman GAX expression cassette utilizing the suicide shuttle pXL3521; itleads to the generation of AV_(1.0)CMV.Gax adenovirus. Plasmid pXL3497derives from pXL2689 (Crouzet et al, Proc Natl Acad Sci USA, 1997, 94:1414-1419) and displays a (Gly-Ser)₅-(Lys)₇ peptide at the C-terminusof the fiber protein. Following Pac1 restriction and transfection inE1-transcomplementing cells, this backbone was used to generate virusAV_(1.k)CMV.lacZ which is identical to AdZ.F(pK7)bgal described byWickham et al (1997, J Virol, 71 :8221-8229).

Production and Quantitation of Adenoviruses

Transfections of adenoviral genomes in PER.C6 cells were performed inthe presence of lipofectAMINE in T25 cm² flasks. Briefly, 5 μg ofPacI-digested DNA plasmid diluted into H₂O were mixed with 23 μl ofLipofectamine. After gentle mixing, the suspension was incubated for 30min at room temperature. In the meantime, the cells at 50-60% confluencewere washed twice with phosphate buffered saline which was then replacedby the LipofectAMINE/DNA mixture to which 3.8 ml of DMEM without serumwere added. The cells were incubated at 37° C. for 5 to 8 hours, afterwhich the medium was replaced by DMEM containing 10% fetal calf serumand 10 mM MgCl₂. Three days after transfection the cells were split intoone T75 cm2 flask. Cells and supernatant were harvested at full CPE (day10-14) and freeze/thawed for three cycles followed by centrifugation andcollection of the supernatant. The EDRAG technology generates ahomogenous (i.e., clonal) population; therefore plaque purification isnot necessary.

Adenoviral particles were precisely quantified by chromatography on aSepharose type support.

PER.C6 cells were infected at a confluence of approximately 70% withrecombinant adenovirus at an MOI between 10 and 100 viral particles percell in T150 cm² flasks. When the cytopathic effect was complete, cellsand supernatant were harvested, freeze/thawed for 3 cycles, centrifugedand the supernatant collected. In certain cases, the recovery processhad to be adapted (see below).

Infectivity of the modified viruses was assessed in vitro in primarycells of various origin (with a special emphasis for smooth muscle cellsand endothelial cells of human origin), and a panel of human andnon-human tumor cell lines that are refractory to infection because theyexpress limiting levels of adenovirus receptor at their cellular surface(see examples 8 to 10). Recombinant knob was used as a competitor whencells easily infectable by Ad5 were used.

Viruses Modified in the HI Loop by Insertion of a Peptide TargetinguPAR.

Residues 538 to 548 of the Ad5 fiber (GTQETGDTTPS) (SEQ ID NO:72) weredeleted and replaced with 6 different peptides flanked with GSS linkers.Construction of the corresponding shuttle plasmids, plasmid backbonesand viruses were carried out as detailed in Example 2. All viruses wereviable, but some (especially viruses AE43, AE44 and AE45) presented analtered stability as compared to their unmodified control virus. It washowever possible to get yields comparable to a control virus (i.e.,10000-20000 VP/cell) by adapting the procedure: PERC6-infected cellswere harvested 3 days post infection and lysed using a mild buffer (Tris10 mM pH7.5, MgCl2 1 mM, Tween 20 1%, NaCl 0.25M) instead of successivefreezing/thawing cycles; viruses were then purified byultracentrifugation on CsCl gradients. TABLE 11 HI loop insertion ofuPAR targeting peptides. adeno- SEQ viral Peptide sequence ID plasmidSelected peptide with linkers NO: pAE42 ATF domain (aa 14 gly-ser-ser-16 to 32 of mature LNGGTCVSNKYFSNIHWCN- human urokinase) gly-ser-serpAE45 mutated ATF do- gly-ser-ser- 17 main (increasedLNGGTAVSNKYFSNIHWCN- affinity for uPAR) gly-ser-ser pAE48 peptideselected gly-ser-ser- 19 by phage-display AEPMPHSLNFSQYLWYT- gly-ser-serpAE46 a mutant of the gly-ser-ser- above selected AEPMPHSLNFSQYLWT- 18peptide gly-ser-ser pAE43 uPAR-binding pep- gly-ser-ser- 20 tide (Vn4)from RGHSRGRNQNSR-gly- human vitronectin ser-ser pAE44 uPAR-binding pep-gly-ser-ser- 21 tide (Vn3) from NQNSRRPSRA- human vitronectingly-ser-ser

Viruses Modified in the HVR5 Loop by Insertion of a Peptide TargetinguPAR.

Residues 269 to 281 of the Ad5 hexon (TTEATAGNGDNLT) (SEQ ID NO:125)were replaced with the above 6 uPAR-binding peptides flanked on bothsides by suitable linkers (gly-ser). Construction of the correspondingplasmid backbones and adenoviruses were carried out as in Example 1. Allviruses were viable. Some of the constructs (e.g., AE27, AE28) displayedsome unstability and were purified accordingly (see above). TABLE 12HVR5 loop insertion of uPAR targeting peptides. adeno- SEQ viral Peptidesequence ID plasmid Selected peptide with linkers NO: pAE26 ATF domaingly-ser-  7 LNGGTCVSNKYFSNIHWCN- gly-ser pAE29 mutated ATF gly-ser-  8domain LNGGTAVSNKYFSNIHWCN- gly-ser pAE47 peptide selected gly-ser- 10by phage-display AEPMPHSLNFSQYLWYT- (Goodson et al., gly-ser 1994,PNAS91: 7129-14 7133 pAE30 a mutant of the gly-ser- above selectedAEPMPHSLNFSQYLWT-  9 peptide gly-ser pAE27 uPAR-binding pep- gly-ser- 11tide (Vn4) from RGHSRGRNQNSR-gly- human vitronectin ser pAE28uPAR-binding pep- gly-ser- 12 tide (Vn3) from NQNSRRPSRA- humanvitronectin gly-ser

Viruses Modified in the HI Loop by Insertion of a Peptide Targeting uPARand Harboring Shortened Fibers.

Residues 538 to 548 of the Ad5 fiber (GTQETGDTTPS) (SEQ ID NO:72) weredeleted and replaced with 6 uPAR-binding peptides flanked on both sidesby suitable linkers (gly-ser-ser) as described above. Thesemodifications were combined with shortened fiber shafts (see Example 3)as summarized in the following table: TABLE 13 Class of short-shaftedviruses modified in the HI loop by insertion of a peptide targeting uPARTail Shaft Knob modification Ad5 tail Ad3 shaft Ad5 HI loop insertionsAd5 tail repeats 1 to 3 and 17 to 22 Ad5 HI loop insertions of Ad5 Ad5tail repeats 1 to 3 and 20 to 22 Ad5 HI loop insertions of Ad5

Viruses Modified in the HVR5 Loop by Insertion of a Peptide TargetinguPAR and Harboring Shortened Fibers.

Residues 269 to 281 of the Ad5 hexon were replaced with 6 differentpeptides flanked by suitable linkers (gly-ser) as described above. Thesemodifications were further combined with the shortening of the fibershaft as described above.

For example, virus AE65 contains the NQNSRRPSRA peptide (SEQ ID NO.6)flanked by gly-ser linkers in place of hexon HVR5 and displays ashortened fiber (shaft deletion encompassing repeats 4 to 16). Anotherexample is virus AE63 which is identical to AE65 except that it containsthe DCRGDCF peptide instead of the Vn3 peptide (see Example 10, FIG.13).

Viruses Modified for Targeting by a C-Terminal Addition of an 8 AminoAcids Linker Followed by a Peptide Targeting uPAR.

The stop codon of the fiber is replaced with the proline codon of thelinker. The linker sequence used for all constructs was PKRARPGS.

The 3 end of the fiber protein coding sequence was modified to introducean FspI site. PCR mutagenesis was used to generate a single basesubstitution (nucleotide 32778) which creates a silent mutationintroducing a novel recognition site for FspI. The Ad5 fiber knob wasamplified from the Ad5 genome using primers MOL1(5′-ggaactttagaaatggagatcttactgaagg-3′) (SEQ ID NO:126) and MOL3(5′-cgattctttattcttgcgcaatgtatgaaaaag-3′) (SEQ ID NO:127).

The primer MOL3 nearly matches the Ad5 nucleotides 32762-32794 with aslight modification resulting in the creation of the FspI restrictionsite. This amplification product was introduced in pCR2.1 (Invitrogen)to create pMA51.

The region downstream from the stop codon of the fiber protein codingsequence was modified by PCR-mutagenesis to introduce AatII, NruI, SpeIrestriction sites upstream the polyA region using the oligonucleotidesMOL2 (5′-cttaagtgagctgcccggggag-3′) (SEQ ID NO:128) and MOL4(5′-ggatccaatgaacttcatcaagt-3′) (SEQ ID NO: 129), and cloned in pCR2.1(Invitrogen) to create pMA52.

The sequence coding for the linker peptide was created by annealing oftwo single-stranded oligonucleotides: MOL7(5′-aattctgcgcaagaaccaaagagggccaggcccggatcctaagacgtct-3′) (SEQ IDNO:130) and MOL8(5′-ctagagacgtcttaggatccgggcctggccctctttggttcttgcgcag-3′) (SEQ IDNO:131). This duplex was cloned between the EcoRI and XbaI sites ofpBSSK+ (Stratagene) creating pMA53.

Finally, the linker sequence was introduced at the 3′-end of the fiberprotein coding sequence by cloning the fragments BglII-FspI from pMA51and FspI-XbaI from pMA53 into the BamHI and XbaI sites of pXL2756 tocreate the vector pMA55.

The shuttle vector pMA56 was constructed by cloning the SmaI-AatIIfragment of pMA52 into pMA55 SmaI-AatII restriction sites. Shuttleplasmid pMA55 was recombined with plasmid backbone pXL3006 in the G4977bacterial strain according to the method described by Crouzet et al.,supra, to obtain the plasmid backbone 22.3 which contains aPacI-excisable E1E3-deleted CMV/lacZ recombinant viral genome encodingfibers with C-terminal modifications.

Further cloning were performed to add the uPAR-targeting ligands at theC-ter of the fiber using a PKRARPGS linker. Briefly, shuttle plasmidsencoding these modifications were constructed and recombined withadenoviral plasmid backbones pXL3091 (RSV-lacZ in place of E1), pXL3006(CMV-lacZ in place of E1) or pXL3527 (hGax expression cassette in placeof E1) in the G4977 bacterial strain according to the EDRAG method togenerate adenoviral backbones displaying lacZ or Gax expressioncassettes and C-terminally modified fiber proteins.

With the exception of bC12x, all expected viruses were recoveredfollowing transfection of PacI-restricted backbones into 911, 293 orPER.C6 cells. Their productivity (VP/cell) was comparable with that oftheir unmodified control virus. TABLE 14 Viruses modified by aC-terminal addition of an 8 amino acids linker followed by a peptidetargeting UPAR. adenoviral SEQ backbones/ Sequence ID viruses Selectedpeptide of the peptide NO: bc9x ATF domain (from LNGGTCVSNKYFSNIHWCN 1(RSV lacZ) aa 14 to 32) bc10x mutated ATF do- LNGGTAVSNKYFSNIHWCN 2 (RSVlacZ) main (from aa 14 to 32) bc12x peptide selected AEPMPHSLNFSQYLWYT 4(RSV lacZ) by phage-display bc11x a mutant of the AEPMPHSLNFSQYLWT 3(RSV lacZ) above selected peptide bc13x vitronectin uPAR- NQNSRRPSRA 6(RSV lacZ) binding domain Vn3 bc14x vitronectin uPAR- RGHSRGRNQNSR 5(RSV lacZ), binding domain bc15x Vn4 (CMV lacZ) and pXL3570 (Gax)

In additional experiments, these modifications were combined withshortening the fiber shaft as examplified above.

Example 5 Specific Targeting of Cells Expressing an αv Integrin Receptor

A high affinity αv integrin binding peptide (CDCRGDCFC refered to asRGD-4C; see also Pasqualini et al., 1997, Nature Biotech. 15:542) and avariant thereof (DCRGDCF refered to as RGD-2C) have been included withinthe fiber HI hexon and the HVR5 loops, or added to the C-terminal end ofthe fiber protein. These viruses contain a heterologous gene (lacZ)inserted in the E1 region, which has been deleted from the viruses

The methods for insertion of these peptides in the hexon HVR5 loop andthe fiber protein HI loop were as described for the poliovirus epitopesas described in Examples 1 and 2, above.

Viruses Modified in the HI Loop by Insertion of a Peptide Targeting αvIntegrins, Associated or not with a Shortened Fiber.

Residues 538 to 548 of the Ad5 fiber were deleted and replaced with thepeptide flanked with GSS linkers. Adenoviral plasmid backbonescontaining a CMVlacZ expression cassette were transfected in PER.C6cells: virus AE60 was viable and could be amplified in PER.C6 cells witha productivity comparable to a control virus, whereas repeatedtransfection of PacI-digested pAE59 DNA did not generate thecorresponding virus, suggesting that this particular construct could notgrow efficiently. TABLE 15 Viruses modified in the HI loop by insertionof a peptide targeting ανintegrins. adeno- SEQ viral Sequence at the IDbackbones Selected peptide peptide with linkers NO: pAE59 CDCRGDCFCgly-ser-ser-CDCRGDCFC 22 (SEQ ID NO:124) -gly-ser-ser pAE60 DCRGDCFgly-ser-ser-DCRGDCF- 23 (SEQ ID NO:148) gly-ser-ser

Viruses modified in the HI loop by insertion of a peptide targeting αvintegrins, associated with a shortened fiber are also obtained.

Viruses Modified in the HVR5 Loop by Insertion of a Peptide Targeting αvIntegrins, Associated or not with a Shortened Fiber.

The amino acids 269 to 281 of the Ad5 hexon were deleted and replacedwith the peptide (the same sequences as described in the table abovewere used) flanked with GS linkers. Adenoviral plasmids containing aLacZ or a hGax expression cassette were transfected in PERC6 or 911cells leading to the recovery of all 3 viruses. A loss of productivitywas observed for AE58 and AE63 viruses in 293 cells, whereas AE57behaved normally: this is likely due to an incorrect folding/stabilityof the hexon in the case of AE58, and to the decrease in Ad receptorbinding of the shortened fiber in the case of AE63. TABLE 16 Virusesmodified in the HVR5 loop by insertion of a peptide targetingανintegrins, associated or not with a shortened fiber Fea- adeno- turesviral Sequence of SEQ of the back- Selected the peptide ID fiber bonespeptide with linkers NO: shaft pAE58 CDCRGDCFC gly-ser-CDCRGD 13 unmod-(SEQ ID NO:124) CFC-gly-ser ified pAE57 DCRGDCF gly-ser-DCRGD 14 unmod-(LacZ) (SEQ ID NO:148) CF-gly-ser ified and pXL3664 (Gax) pAE63 DCRGDCFgly-ser-DCRGD 14 short- (SEQ ID NO:148) CF-gly-ser ened (re-peats 1-3and 17-22 of Ad5)

The inclusion of the RGD-2C peptide in the hexon was also combined withthe addition of the linker-peptide sequence PKRARPGS-K7 (SEQ ID NO.132)at the C-terminus of the fiber. The corresponding virus was viable.

Example 6 Construction of Heparan Sulfate Proteoglycans Targeted Viruses

Fiber-modified adenoviruses containing a lacZ or Gax expression cassettewere constructed by genetic modification of the adenoviral genome in E.coli using the EDRAG technology and produced in 911 or PER.C6 cells (seeMaterial and Methods of Example 4).

Three ligands expected to bind to heparan sulfate proteoglycans wereidentified: the heptalysine stretch (K7) described by Wickham et al.(1997, J Virol, 71 :8221-8229), the arginine-leucine repeated motifRRLLRRLLRR (SEQ ID NO.133), described in patent application WO95/21931,and the peptide fragment from FGF-1 binding to heparin KRGPRTHYGQK (SEQID NO.134) described by Digabriele et al (1998, Science, 393 :812-817).

Among others, five viruses have the heptalysine K7 stretch at theC-terminus of the fiber and differ by the transgene (lacZ or Gax) and/orthe identity of the connecting (no linker, (GS)5 or PKRARPGS). Virus3497 was included as a reference (Wickham et al., 1997, J Virol, 71:8221-8229). The importance of the linker and/or peptide in terms ofviral production and transduction efficacy in vitro and in vivo was thenassessed.

A polylysine stretch has also been included within the hexon HVR5 or thefiber HI loops in viruses containing a heterologous gene (lacZ) insertedin the E1 region TABLE 17 Heparan Sulfate Proteoglycans Targeted virusesadenoviral backbones Modification of the capsid pAE61 Substitution ofhexon aa 269-281 with GS-K5-GS (SEQ ID N^(o) 135) pAE62 Substitution offiber aa 538-548 with GSS-K7- GSS (SEQ ID N^(o) 136) pXL3497 (lacZ) and(GS)5-K7 (SEQ ID N^(o) 137) added to the fiber pXL3528 (gax) C-terminuspXL3496 (lacZ) and PKRARPGS-K7 (SEQ ID N^(o) 138) added to the pXL3569(gax) fiber C-terminus pXL3631 (lacZ) K7 (SEQ ID N^(o) 139) added to thefiber C-terminus pXL3662 (lacZ) and PKRARPGS-KRGPRTHYGQK (SEQ ID N^(o)140) pXL3665 (gax) added to the fiber C-terminus pXL3663 (lacZ) andPKRARPGS-RRLLRRLLRR (SEQ ID N^(o) 141) pXL3666 (gax) added to the fiberC-terminus

All these viruses were viable and could be amplified in E1transcomplementing cells.

The following table 18 summarizes the yield obtained in one experimentperformed in PER.6 cells after infection of the cells at moi 10 to 100VP/cell. TABLE 18 Amplification of viruses in PER.C6 cells Virus Viralparticles/cell 3497 1200 3528 2300 3496 2700 3569 1000 3631 6000 366212160

Productivity was differently affected by these C-terminal fiberextensions. Overall, these and other data indicate that the presence andidentity of the connecting linker sequence added to the fiber C-terminusgreatly influences the adenovirus infection cycle/behavior.

For a given linker sequence, the identity/nature of the foreign peptideper se was also found to be an important parameter. For example, theconstruct with the RRLLRRLLRR peptide (AV_(1s)CMV.lacZ or Ad3663)yielded very low titers whereas its replacement by the KRGPRTHYGQKpeptide (AV_(1.f)CMV.lacZ or Ad3662) restored productivity.

The recovery process had also to be optimized for most of these viruses.For example, a total of 5.10¹² VP of Ad3497 was successfully purified bya two-step chromatography procedure and finally resuspended in Tris 20mM pH8.4-10% glycerol, with an overall particle recovery of 68% asdescribed hereabove.

Example 7 Construction of Viruses with a Vn4 Peptide within the HI Loop

As mentionned in example 4, the AE43 virus somehow displayed some levelsof is unstability that required an optimized recovery process. To rescueits stability without loosing its advantageous binding characteristics,the Vn4 peptide was introduced in the HI loop in various neighboringcontexts (see following table 19). The corresponding viruses (whichcontained a lacZ or Gax expression cassette in place of E1) wereconstructed by recombinational cloning in E. coli and amplified in 911or PERC6 cells as described in Example 4. TABLE 19 viruses with a Vn4peptide in the HI loop SEQ ID virus Modification of the capsid N^(o)AE43 Substitution of fiber aa 538-548 with GSS- 20 Vn4-GSS GL11Substitution of fiber aa 538-548 with GSS- 143 Vn4+Vn3-GSS* GL12Substitution of fiber aa 538-548 with GTSE- 144 Vn4-GSS GL13Substitution of fiber aa 538-548 with GTQE- 145 Vn4-GSS GL14Substitution of fiber aa 538-548 with GSSS- 146 Vn4-GSS GL16Substitution of fiber aa 538-548 with GSS- 147 Vn4-GGS GL17 Substitutionoffiber aa 541-548 with SS- 142 Vn4-GSS 3630 (lacZ) and Insertion ofSS-Vn4-GS between fiber aa 150 3629 (Gax) 546 (Thr) and 547 (Pro)*Vn4+Vn3 = RGHSRGRNQNSRRPSRA is derived from human vitronectin

Almost all constructs exhibited a productivity that was comparable tothat of their unmodified control virus (see following table 20): TABLE20 Virus Viral particles/cell control virus 20000 (n = 2) AE43 20000 (n= 2) GL11  200 (n = 1) GL12 20000 (n = 3) GL13 25000 (n = 2) GL14 20000(n = 1) GL16  4000 (n = 2) GL17 15000 (n = 3) 3630 10000 (n = 3) 362914000 (n = 1)

Virus stability was also differently affected by these modifications. Inparticular, some of them (e.g., AE43, GL11, GL14 and GL16) weresensitive to successive rounds of freezing/thawing so the infected cellshad to be lysed in mild conditions (Tris 10 mM pH 7.5, MgCl2 1 mM, Tween20 1%, NaCl 0.25M) for recovery, again emphasizing the influence of thelinker sequences on the virus behavior (see also Example 9, FIG. 12).

Example 8 Evaluation of Targeted Viruses in Human Primary Cells

Materials and Methods

Cell Culture

Primary cultures of rat and rabbit smooth muscle cells were preparedfrom thoracic aortas of adult male Spraggue-Dawley rats or of adult NewZealand White rabbits according to Mader et al (1992, J Gerontol. Biol.Sci. 47: b32-b36). Cells were propagated at 37° C. in Dulbecco'smodified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS)and penicillin/streptomycin. Human aorta smooth muscle cells and HUVECwere purchased from Clonetics and cultured following instructions of themanufacturer.

Adenoviral Mediated In Vitro Gene Transfer and Gene Expression Assays

Cells were seeded onto 24-well or 12-well plates one to two days priorto experiments. Cells were infected at moi 100 or 1000 (VP/cell) byincubation of the adenoviral vector diluted in serum free culture mediumfor one hour at 37° C. Cells were then washed and growth medium wasadded for 48 hours to allow β-galactosidase expression. The cells werethen lysed and assayed for β-galactosidase activity by using Luminescentβ-galactosidase genetic reporter system II (Clontech), or Xgal stained.

Results

Construction Encoding β Galactosidase Reporter Gene

Data presented in the two following tables 21 and 22 show that mostviruses are able to transduce hSMC more efficiently than a controlvirus, and that viruses AE30, AE42, AE43, AE44, AE45, AE57, AE58, AE61,AE62, BC15X and 3497 are good candidates for further in vitro and invivo studies. Experiments performed on primary SMCs infected at an moiof 1000 VP/cell at different passage demonstrated a very significantgain of transduction for several of the modified viruses (examples areprovided in tables 21 and 22). Extracts were prepared 48 hrpost-infection at which time protein and β-galactosidase activity werequantified. Transduction efficacy was then assessed by comparing thelevels of lacZ specific activity (RLU/protein extract). TABLE 21Experiment #1 Transduction efficacy in Virus human SMC control 1 AE303.5 AE43 53 AE44 23 AE45 10 3497 115 BC15X 26

TABLE 22 Experiment #2 Transduction efficacy in Virus human SMC control1 AE27 16 AE29 6 AE30 133 AE42 95 AE43 825 AE48 31 AE57 846 AE58 264AE60 52 AE61 781 AE62 772 BC15X 47

The following table 23 summarizes the data obtained in SMC fromdifferent species: most viruses were able to efficiently transducenon-human cells, indicating that there is no species barrier in theirentry pathway. TABLE 23 infection of SMC from different speciesTransduction efficacy Transduction efficacy Transduction efficacy Virusin rat SMC in pig SMC in rabbit SMC control 1 1 1 BC15X —  97 (n = 1) —AE 43  77 (n = 2)  753 (n = 2) 131 (n = 3) AE 62 166 (n = 2) 2166 (n =2) 320 (n = 5) 3497  50 (n = 2)  349 (n = 2)  55 (n = 2) 3496 295 (n =2) 2318 (n = 2) 437 (n = 2) AE 57 — 2960 (n = 1) — AE 63  21 (n = 2) 293 (n = 2)  23 (n = 2)n = number of experiments

To demonstrate that the increase of transduction observed followingcapsid modification was due at least in part to an increase ininfectivity, quantitative PCR was carried out on viral DNA extractedfrom infected human SMC at moi 1000 VP/cell. Simultaneously, RLUmeasurements were performed on protein extracts.

Table 24 indicates that the gain in human smooth muscle cellstransduction efficiency that caracterized the best candidate virusescorrelated in all cases (although to different extents) with an increasein DNA entry. The correlation was especially good for viruses AE43 andBC15X. TABLE 24 genome delivery and transgene expression RLU/μg virusGenomes/cell^(a) total protein Ratio RLU Ratio genomes control 3.3191641 1 1 AE30 211 677345 3.5 64 AE43 87 10207798 53 26.5 AE45 4271851206 10 130 3497 124 22115125 115 38 BC15X 40 5004672 26 12.3^(a)genomes/cell is the ratio between the amount of viral genomes asquantified by PCR and the number of infected cells.^(b)ratio RLU: ratio between the RLU level of the indicated virus andthe RLU level of the control virus^(c)ratio genomes: ratio between the genomes amount of the indicatedvirus and the genomes amount of the control virus.

The entry pathway in SMC was also analyzed in competition with solubleheparin or soluble uPAR as illustrated in FIGS. 4 and 5.

In FIG. 4, hSMC were infected at moi 1000 (VP/cell) in presence ofincreasing doses of soluble heparin. Cells were washed in PBS beforefurther incubation at 37° C. Extracts were prepared 48 hr post-infectionat which time total proteins and β-galactosidase activity werequantified. The data show that infection of hSMC bypolylysine-containing viruses is specifically inhibited by solubleheparin showing that these viruses likely bind to cellular heparansulfate proteoglycans at the cell surface. In contrast, and as expected,AE43 does not use this particular pathway for entry.

In FIG. 5, viruses were preincubated with increasing doses of solubleuPAR (0 to 9, 4 μg/ml) before incubation on hSMC at moi 1000 (VP/cell).Cells were washed in PBS before further incubation at 37°. Extracts wereprepared 48 hr post-infection at which time total proteins andβ-galactosidase activity were quantified. The data show that infectionof hSMC by some of the uPAR-targeting viruses (e.g., AE43) isspecifically inhibited by recombinant soluble uPAR. That hSMC expressuPAR at their cellular surface (data not shown) strongly suggests theuse of this particular receptor for cellular entry.

Gax Encoding Targeted Adenoviruses

Gax expression level in infected hSMC was analyzed by Western blot(following figures). The highest increase in Gax expression was obtainedafter infection with virus 3528 (K7 at the C-terminus of the fiber). Gaxprotein was detected with all modified viruses at moi 3000 VP/cell,whereas Gax expression in cells infected with the control virus at thesame moi was undetectable.

FIG. 6 illustrates the Gax expression in human SMC infected withtargeted adenovirus. Protein extracts were prepared 24 hours afterinfection at the indicated moi (VP/cell). (1) AV_(1.0)CMVrGax moi 3.10⁴,(2) AV_(1.0)CMVrGax moi 10⁴, (3) 3528 moi 10⁴, (4) 3528 moi 3.10³, (5)3528 moi 300, (6) 3569 moi 3.10³, (7) 3569 moi 300, (8) 3570 moi 10⁴ and(9) 3570 moi 3.10³.

FIG. 7 ilustrates the Gax expression after human SMC infection withtargeted adenovirus. Protein extracts were prepared 24 hours afterinfection at the indicated moi (VP/cell).

Left panel: (1) AV_(1.0)CMVrGax moi 3.10⁴ (2) AV_(1.0)CMVhGax moi 3.10⁴,(3) AV_(1.0)CMVhGax moi 3.10³, (4) 3528 moi 3000, (5) 3528 moi 300, (6)3528 moi 30, (7) 3569 moi 3000, (8) 3569 moi 300, (9) 3569 moi 30

Right panel: (1) AV_(1.0)CMVrGax moi 3.10⁴ (2) AV_(1.0)CMVhGax moi3.10⁴, (3) AV_(1.0)CMVhGax moi 3.10³, (4) 3570 moi 3000, (5) 3570 moi300, (6) 3570 moi 30, (7) 3629 moi 3000, (8) 3629 moi 300, (9) 3629 moi30

Gax expression was also evidenced by FACS analysis after infection ofhSMC at different moi. Even if the sensitivity of this technique is muchhigher than Western blotting, the results correlate with Westernexperiments and show the relative superiority of viruses 3528 and 3569over 3570 and 3629 in their ability to efficiently transduce hSMC, asilustrates on the following table 25. TABLE 25 FACS analysis of Gaxexpression after infection of human SMC.(% of Gax expressing cells wasdetermined 24 hours after infection.) Virus 10000 VP/cell 1000 VP/cell100 VP/cell AV_(1.0)CMVrGax 100% 52% — AV_(1.0)CMVhGax  86%  8% — 3528nd 99% 99% 3569 nd 99% 99% 3570 nd 99%  8% 3629 nd 99% 18%

Example 9 In Vitro Evaluation of Targeted Viruses in Human Tumoral Cells

Hs578T cells (human breast tumor cells) are quire refractory to Ad5infection most likely because they express limiting amounts of the virusreceptor at their cellular surface. In practice, an moi as high as 10⁵VP/cell is necessary to infect 50% of the cells. They were tested fortheir ability to be transduced by a panel of capsid-modified vectors.

FIGS. 8A, 8B, 8C and 9 illustrate infection of Hs578T with differenttargeted viruses. Cell extracts were prepared 48 h post infection. Thedata show that AE43, AE44, AE45 or 3497 are very efficient intransducing this cell type.

The pathway of infection of AE43 was analyzed by competition withsoluble knob fiber or soluble uPAR (FIGS. 10 and 11).

In FIG. 10, AE43 was preincubated with soluble uPAR before infection ofHs578T. Cell extracts were prepared 48 h post infection.

In FIG. 11, AE43 was preincubated with soluble UPAR (10 μg/2 10⁸ VP) orsoluble knob (100 μg/ml) before infection of Hs578T. Cell extracts wereprepared 48 h post infection.

The results indicate that AE43 does not enter the cell via the classicalknob-CAR pathway but rather uses a uPAR-dependent pathway for entry.

Finally, Vn4-containing viruses 3630, GL12, GL14 or GL17 were comparedto AE43 in their ability to transduce Hs578T cells. As shown in FIG. 12at 48 h post infection, the nature of the connecting linkers indeed cangreatly influence the efficacy with which a binding peptide inserted inthe HI loop interacts with its specific receptor at the cellularsurface.

Example 10 In Vitro Evaluation of Targeted Viruses in Murine TumoralCells

Murine NIH-3T3 cells are very resistant to Ad5 infection as more than100000 VP/cell is necessary to infect 50% of the cells. They were testedfor their ability to be transduced by a panel of capsid-modifiedviruses. Cell extracts were prepared 48 h post infection. FIG. 13include data from a representative experiment which demonstrate thatAE28, AE43, AE44, AE58, AE57 or AE62 are particularly efficient.

Also, and importantly, AE63 (short fiber, RGD-2C in hexon) was shown tobe partially able to rescue the defect associated with its short-shaftedcontrol virus (vBS1; no insertion in hexon). Capsid modifications thatimpair the native entry pathway (e.g., fibers displaying short shafts)can therefore be combined with capsid modifications that provide anadditional, CAR-independent, pathway for infection.

The pathway of infection of AE43 and BC15X was analyzed by competitionwith soluble UPAR (FIGS. 14A and 14B). In FIGS. 14A and 14B, virusesBC15X (A) and AE43 (B) were preincubated with increasing doses ofsoluble uPAR before incubation with cells (moi 1000 and 200 VP/cell,respectively). Cells were then washed and further incubated at 37° C.for 48 h before preparation of cell extracts. These and other dataindicate that these viruses use uPAR for infection.

Example 11 In Vivo Evaluation of Targeted Viruses in a Restenosis RabbitModel

The in vivo evaluation of some of the targeted viruses was performed ina an atheromatous double injury rabbit model, which is a good model forrestenosis: transfer takes place in atheromatous iliac arteries; rabbitsare fed 120 g daily of 1% cholesterol diet and at 3 weeks a first injuryby balloon angioplasty is performed with a 2.5 mm diameter Nycomedballoon catheter. One week later, adenoviral gene transfer is performed.

Microscopic quantification of SMC staining for β-galactosidase was usedto define the efficacy of gene transfer (histochemical analysis).Briefly, 32 sections/artery were examined and XGal-positive cells werecounted. The data are presented as the highest score among the 32sections for one artery. Results are presented in the following table26. TABLE 26 In vivo evaluation of targeted viruses in a restenosisrabbit model Virus injected 10¹¹ VP/artery 5 · 10¹¹ VP/artery Controlvirus positive arteries: 0/8 positive arteries: 7/10 6 arteries with <30stained cells 1 artery with 200-400 stained cells AE57 positivearteries: 0/6 positive arteries: 2/2 RGD-2C in hexon 2 arteries with <30stained cells AE43 positive arteries: 5/6 VN4 in HI fiber 2 arterieswith <30 stained cells 2 arteries with 30-100 stained cells 1 arterywith >400 stained cells BC15X positive arteries: 4/4 VN4 1 artery with<30 stained cells at C-ter fiber 1 artery with 30-100 stained cells 1artery with 100-200 stained cells 1 artery with 200-400 stained cells

These data indicate that adenoviruses AE43 and BC15X transduce arterialwall with a dramatically increased efficacy as compared to theirunmodified control.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An adenovirus comprising a hexon HRV5 loop from which at least a partof the hexon HRV5 loop is replaced with a binding peptide, or targetingsequence, flanked by connecting amino acid spacers so as to functionallydisplay its binding specificity at the capsid surface.
 2. The adenovirusaccording to claim 1 wherein about 6 to 17 amino acids of the hexon HVR5loop are replaced.
 3. The adenovirus according to claim 2 wherein nomore than 14 amino acids of the hexon HVR5 loop are replaced.
 4. Theadenovirus according to claim 3 wherein about 13 amino acids from thehexon HVR5 loop corresponding to about amino acid residue 269 to aboutamino acid residue 281 of adenovirus serotype 5 (Ad5) are replaced. 5.The adenovirus according to claim 4, wherein the spacers comprise anamino acid selected from the group consisting of glycine, serine,threonine, alanine, cysteine, aspartate, asparagine, methionine andproline.
 6. The adenovirus according to claim 5, wherein the spacerscomprise an amino acid selected from the group consisting of glycine andserine.
 7. The adenovirus according to claim 5, wherein the first aminoacid in at least one of the spacers is an amino acid selected from thegroup consisting of glycine, serine, threonine, alanine, cysteine,aspartate, asparagine, methionine and proline.
 8. The adenovirusaccording to claim 7, wherein the first amino acid in the spacers is anamino acid selected from the group consisting of glycine, serine,threonine, alanine, cysteine, aspartate, asparagine, methionine andproline.
 9. The adenovirus according to claim 8, wherein the first aminoacid in at least one of the spacers is a glycine residue.
 10. Theadenovirus according to claim 8, wherein the first amino acid of thespacers is a glycine residue.
 11. The adenovirus according to claim 5wherein at least one of the spacers is a dipeptide.
 12. The adenovirusaccording to claim 11 wherein at least one of the spacers is a Gly-Serdipeptide.
 13. The adenovirus according to claim 4, wherein thetargeting sequence is a ligand epitope for a urokinase-type plasminogenactivator receptor (UPAR).
 14. The adenovirus according to claim 13wherein the targeting sequence is selected from the group consisting ofLNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2);AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4);RGHSRGRNQNSR (SEQ ID NO: 5); and NQNSRRPSRA (SEQ ID NO: 6).
 15. Theadenovirus according to claim 12 wherein the targeting sequence,including the spacers, is selected from the group consisting of: (SEQ IDNO: 7); A. gly-ser-LNGGTCVSNKYFSNIHWCN-gly-ser; (SEQ ID NO: 8) B.gly-ser-LNGGTAVSNKYFSNIHWCN-gly-ser; (SEQ ID NO: 9) C.gly-ser-AEPMPHSLNFSQYLWT-gly-ser; (SEQ ID NO: 10); D.gly-ser-AEPMPHSLNFSQYLWYT-gly-ser; (SEQ ID NO: 11) E.gly-ser-RGHSRGRNQNSR-gly-ser; (SEQ ID NO: 12) F.gly-ser-NQNSRRPSRA-gly-ser; (SEQ ID NO: 13) G.gly-ser-CDCRGDCFC-gly-ser; (SEQ ID NO: 14) H. gly-ser-DCRGDCF-gly-ser;and (SEQ ID NO: 15) I. gly-ser-KKKKKKK-gly-ser.


16. The adenovirus according to claim 4 which is derived from humanadenovirus serotype.
 17. The adenovirus according to claim 16 which isderived from human adenovirus subgroup C.
 18. The adenovirus accordingto claim 17 which is derived from human adenovirus serotype
 5. 19. Theadenovirus according to claim 4, wherein said adenovirus comprises afiber protein comprising a fiber shaft, wherein said fiber shaft ismodified to be shorter than a wild-type fiber shaft.
 20. The adenovirusaccording to claim 19 wherein said fiber shaft has been shortened by anin-frame deletion.
 21. The adenovirus according to claim 19 wherein saidfiber shaft has been shortened by replacement with a shaft from anotherserotype.
 22. The adenovirus according to claim 19 wherein said fibershaft is from human subgroup C (Ad2 or Ad5) and has been shortened byreplacement with a shaft from serotype 3 (Ad3)
 23. The adenovirusaccording to claim 19 wherein said fiber shaft contains repeats 1 to 3and 17 to 22 of Ad5; repeats 1 to 3 and 20 to 22 of Ad5; or anadenovirus serotype 3 (Ad3) shaft.
 24. An adenovirus comprising a hexonHI loop from which at least a part of the hexon HI loop is replaced witha targeting sequence, flanked by connecting amino acid spacers so as tofunctionally display the targeting sequence's binding specificity at thecapsid surface.
 25. The adenovirus according to claim 24 wherein about 6to 17 amino acids from the hexon HI loop are replaced.
 26. Theadenovirus according to claim 25, wherein no more than 11 amino acidsfrom the hexon HI loop are replaced.
 27. The adenovirus according toclaim 26 wherein about 11 amino acids from the hexon HI loopcorresponding to about amino acid residue 538 to about amino acidresidue 548 of adenovirus serotype 5 (Ad5) are replaced.
 28. Theadenovirus according to claim 27, wherein the spacers comprise an aminoacid selected from the group consisting of glycine; serine, threonine,alanine, cysteine, aspartate, asparagine, methionine and proline. 29.The adenovirus according to claim 28, wherein the spacers comprise anamino acid selected from the group consisting of glycine and serine. 30.The adenovirus according to claim 27, wherein the first amino acid in atleast one of the spacers is an amino acid selected from the groupconsisting of glycine, serine, threonine, alanine, cysteine, aspartate,asparagine, methionine and proline.
 31. The adenovirus according toclaim 30, wherein the first amino acid in the spacers is an amino acidselected from the group consisting of glycine, serine, threonine,alanine, cysteine, aspartate, asparagine, methionine and proline. 32.The adenovirus according to claim 31, wherein the first amino acid in atleast one of the spacers is a glycine residue.
 33. The adenovirusaccording to claim 32, wherein the first amino acid of the spacers is aglycine residue.
 34. The adenovirus according to claim 27 wherein atleast one of the spacers is a tripeptide.
 35. The adenovirus accordingto claim 34 wherein the at least one of the spacers is a Gly-Ser-Sertripeptide.
 36. The adenovirus according to claim 27, wherein thetargeting sequence is a ligand epitope for a urokinase-type plasminogenactivator receptor (UPAR).
 37. The adenovirus according to claim 36wherein the targeting sequence is selected from the group consisting ofLNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2);AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4);RGHSRGRNQNSR (SEQ ID NO: 5); and NQNSRRPSRA (SEQ ID NO: 6).
 38. Theadenovirus according to claim 27 wherein the targeting sequence,including the spacers, is selected from the group consisting of: A.gly-ser-ser-LNGGTCVSNKYFSNIHWC (SEQ ID NO: 16) N-gly-ser-ser; B.gly-ser-ser-LNGGTAVSNKYFSNIHWC (SEQ ID NO: 17) N-gly-ser-ser; C.gly-ser-ser-AEPMPHSLNFSQYLWT- (SEQ ID NO: 18) gly-ser-ser; D.gly-ser-ser-AEPMPHSLNFSQYLWYT- (SEQ ID NO: 19) gly-ser-ser; E.gly-ser-ser-RGHSRGRNQNSR-gly- (SEQ ID NO: 20) ser-ser; F.gly-ser-ser-NQNSRRPSRA-gly- (SEQ ID NO: 21) ser-ser; G.gly-ser-ser-CDCRGDCFC-gly- (SEQ ID NO: 22) ser-ser; H.gly-ser-ser-DCRGDCF-gly- (SEQ ID NO: 23) ser-ser; and I.gly-ser-ser-KKKKKKK-gly- (SEQ ID NO: 24) ser-ser; J.ser-ser-RGHSRGRNQNSRRPSRA- (SEQ ID NO: 143) gly-ser; K.tyr-ser-glu-RGHSRGRNQNSR- (SEQ ID NO: 144) gly-ser; L.tyr-gln-glu-RGHSRGRNQNSR- (SEQ ID NO: 145) gly-ser; M.ser-ser-ser-RGHSRGRNQNSR- (SEQ ID NO: 146) gly-ser; and N.ser-ser-RGHSRGRNQNSR-gly-gly. (SEQ ID NO: 147)


39. The adenovirus according to claim 27 which is derived from humanadenovirus serotype.
 40. The adenovirus according to claim 39 which isderived from human adenovirus subgroup C.
 41. The adenovirus accordingto claim 40 which is derived from human adenovirus serotype
 5. 42. Theadenovirus according to claim 27 wherein said adenovirus comprises afiber protein comprising a fiber shaft, wherein said fiber shaft ismodified to be shorter than a wild-type fiber shaft.
 43. The adenovirusaccording to claim 42 wherein said fiber shaft has been shortened by anin-frame deletion.
 44. The adenovirus according to claim 42 wherein saidfiber shaft has been shortened by replacing it with the shaft fromanother serotype
 45. The adenovirus according to claim 44 wherein thefiber shaft is from human subgroup C (Ad2 or Ad5) and has been shortenedby replacing it with the shaft from serotype 3 (Ad3)
 46. The adenovirusaccording to claim 42 wherein the fiber shaft contains repeats 1 to 3and 17 to 22 of Ad5; repeats 1 to 3 and 20 to 22 of Ad5; or anadenovirus serotype 3 (Ad3) shaft
 47. A recombinant adenoviruscomprising a fiber protein wherein a binding peptide, or targetingsequence, is connected to the C-terminus of the fiber protein by aconnecting spacer, or linker, so as to functionally display its bindingspecificity at the capsid surface.
 48. The recombinant adenovirusaccording to claim 47 wherein the connecting spacer comprise an aminoacid selected from the group consisting of glycine, serine, threonine,alanine, cysteine, aspartate, asparagine, methionine and proline. 49.The recombinant adenovirus according to claim 48 wherein the first aminoacid in the spacer is a proline.
 50. The recombinant adenovirusaccording to claim 47 which is derived from a human adenovirus serotype.51. The recombinant adenovirus according to claim 50 which is derivedfrom human adenovirus subgroup C.
 52. The recombinant adenovirusaccording to claim 50 which is derived from human adenovirus serotype 5.53. The recombinant adenovirus according to claim 47 wherein the fiberprotein is modified to have a fiber shaft that is shorter than awild-type fiber shaft.
 54. The recombinant adenovirus according to claim53 wherein said fiber shaft has been shortened by an in-frame deletion.55. The recombinant adenovirus according to claim 53 wherein said fibershaft has been shortened by replacing it with the shaft from anotherserotype.
 56. The recombinant adenovirus according to claim 54 whereinsaid fiber shaft is from subgroup C and comprises an in-frame deletionencompassing repeats 4 to 16 or repeats 4 to
 19. 57. The recombinantadenovirus according to claim 53 wherein said fiber shaft is fromsubgroup C and has been shortened by replacing it with the shaft fromserotype 3 (Ad3).
 58. The adenovirus according to claim 57 comprising aspacer or linker peptide comprising from 5 to 30 amino acids.
 59. Theadenovirus according to claim 58, wherein the spacer or linker peptidecomprises the sequence PKRARPGS (SEQ ID NO:149).
 60. The adenovirusaccording to claim 59, wherein the targeting sequence is a ligandepitope for a urokinase-type plasminogen activator receptor (UPAR). 61.The adenovirus according to claim 59, wherein the targeting sequence isa peptide fragment from FGF-1 binding to heparin, comprising between 7and 15 amino acids.
 62. The adenovirus according to claim 59, whereinthe targeting sequence is composed of 5 to 10 lysine residues.
 63. Theadenovirus according to claim 59, wherein the targeting sequence iscomposed of almost 7 lysine residues.
 64. The adenovirus according toclaim 59, wherein the targeting sequence is composed of between 5 and 10Arg-Arg and Leu-Leu motifs.
 65. The adenovirus according to claim 59,wherein the targeting sequence is selected from the group consisting ofLNGGTCVSNKYFSNIHWCN (SEQ ID NO: 1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2);AEPMPHSLNFSQYLWT (SEQ ID NO: 3); AEPMPHSLNFSQYLWYT (SEQ ID NO: 4);RGHSRGRNQNSR (SEQ ID NO: 5); NQNSRRPSRA (SEQ ID NO: 6); RRLLRRLLRR (SEQID NO: 133); and KRGPRTHYGQK (SEQ ID NO: 134);
 66. The adenovirusaccording to claim 59, wherein the targeting sequence including thelinker peptide comprises the sequence PKRARPGSKKKKKKK (SEQ ID NO:132).67. The adenovirus according to claim 59, wherein the targeting sequenceincluding the linker peptide comprises the sequence PKRARPGSRRLLRRLLRR(SEQ ID NO:141).
 68. The adenovirus according to claim 59, wherein thetargeting sequence including the linker peptide comprises the sequencePKRARPGSKRGPRTHYGQK (SEQ ID NO:140).
 69. A method for modifying thecellular tropism of an adenovirus vector, comprising A. deleting anative amino acid sequence from a site in a capsid protein of theadenovirus; and B. inserting a targeting peptide sequence connected by afirst spacer at the N-terminus and a second spacer at the C-terminus ofthe targeting sequence; and wherein the targeting peptide is inserted ina deletion site selected from the group consisting of about 13 aminoacids from the hexon HVR5 loop corresponding to about amino acid residue269 to about amino acid residue 281 of adenovirus Ad5; and about 11amino acids from the fiber protein HI loop corresponding to about aminoacid residue 538 to about amino acid residue 548 of Ad5.
 70. The methodaccording to claim 69, wherein the first spacer comprises an amino acidselected from the group consisting of glycine and serine.
 71. The methodaccording to claim 69, wherein the second spacer comprises an amino acidselected from the group consisting of glycine and serine.
 72. The methodaccording to claim 69, wherein the targeting sequence is a ligandepitope for a urokinase-type plasminogen activator receptor (UPAR). 73.The method according to claim 72, wherein the targeting sequence isselected from the group consisting of LNGGTCVSNKYFSNIHWCN (SEQ ID NO:1); LNGGTAVSNKYFSNIHWCN (SEQ ID NO: 2); AEPMPHSLNFSQYLWT (SEQ ID NO: 3);AEPMPHSLNFSQYLWYT (SEQ ID NO: 4); RGHSRGRNQNSR (SEQ ID NO: 5); andNQNSRRPSRA (SEQ ID NO: 6).
 74. The method according to claim 69, whereintargeting sequence, including the spacers, is inserted in the HVR5 loopand is selected from the group consisting of: (SEQ ID NO: 7) A.gly-ser-LNGGTCVSNKYFSNIHWCN-gly-ser; (SEQ ID NO: 8) B.gly-ser-LNGGTAVSNKYFSNIHWCN-gly-ser; (SEQ ID NO: 9) C.gly-ser-AEPMPHSLNFSQYLWT-gly-ser; (SEQ ID NO: 10) D.gly-ser-AEPMPHSLNESQYLWYT-gly-ser; (SEQ ID NO: 11) E.gly-ser-RGHSRGRNQNSR-gly-ser; (SEQ ID NO: 12) F.gly-ser-NQNSRRPSRA-gly-ser; (SEQ ID NO: 13) G.gly-ser-CDCRGDCFC-gly-ser; (SEQ ID NO: 14) H. gly-ser-DCRGDCF-gly-ser;and (SEQ ID NO: 15) I. gly-ser-KKKKKKiK-gly-ser


75. The method according to claim 69, wherein the targeting sequence,including the spacers, is inserted in the fiber protein HI loop and isselected from the group consisting of: A. gly-ser-ser-LNGGTCVSNKYFSNIHWC(SEQ ID NO: 16) N-gly-ser-ser; B. gly-ser-ser-LNGGTAVSNKYFSNIHWC (SEQ IDNO: 17) N-gly-ser-ser; C. gly-ser-ser-AEPMPHSLNFSQYLWT- (SEQ ID NO: 18)gly-ser-ser; D. gly-ser-ser-AEPMPHSLNFSQYLWYT- (SEQ ID NO: 19)gly-ser-ser; E. gly-ser-ser-RGHSRGRNQNSR-gly- (SEQ ID NO: 20) ser-ser;F. gly-ser-ser-NQNSRRPSRA-gly- (SEQ ID NO: 21) ser-ser; G.gly-ser-ser-CDCRGDCFC-gly- (SEQ ID NO: 22) ser-ser; H.gly-ser-ser-DCRGDCF-gly- (SEQ ID NO: 23) ser-ser; and I.gly-ser-ser-KKKKKKK-gly- (SEQ ID NO: 24) ser-ser; J.ser-ser-RGHSRGRNQNSRRPSRA- (SEQ ID NO: 143) gly-ser; K.tyr-ser-glu-RGHSRGRNQNSR- (SEQ ID NO: 144) gly-ser; L.tyr-gln-glu-RGHSRGRNQNSR- (SEQ ID NO: 145) gly-ser; M.ser-ser-ser-RGHSRGRNQNSR- (SEQ ID NO: 146) gly-ser; and N.ser-ser-RGHSRGRNQNSR-gly-gly. (SEQ ID NO: 147)


76. The method according to claim 69, further comprising shortening thefiber protein shaft.
 77. The method according to claim 69, wherein thefiber shaft comprises repeats 1 to 3 and 17 to 22 of Ad5; repeats 1 to 3and 20 to 22 of Ad5; or an Ad3 shaft.
 78. An adenovirus hexon comprisinga deletion of about 13 amino acids from the HVR5 loop corresponding toabout amino acid residue 269 to about amino acid residue 281 ofadenovirus serotype 5 (Ad5) and an insertion at the site of the deletionof a targeting peptide sequence connected by a first spacer at theN-terminus and a second spacer at the C-terminus of the targetingpeptide sequence.
 79. An adenovirus hexon protein comprising a deletionof about 11 amino acids from the HI loop corresponding to about aminoacid residue 538 to about amino acid residue 548 of adenovirus serotype5 (Ad5) and an insertion at the site of the deletion of a targetingpeptide sequence connected by a first spacer at the N-terminus and asecond spacer at the C-terminus of the targeting peptide sequence. 80.An adenovirus fiber protein comprising a linker peptide and a targetingpeptide at its C-terminus.
 81. A method for preferentially expressing agene in a target cell comprising contacting a population of cellscontaining the target cell with an adenovirus of claim 1, wherein thetargeting sequence is a ligand epitope for a receptor on the targetcell.
 82. A method for preferentially expressing a gene in a target cellthat expresses a UPAR comprising contacting a population of cellscontaining the target cell with a targeted adenovirus vector of claim13.
 83. The method according to claim 81, wherein the targetedadenovirus vector comprises a heterologous gene encoding a gene fortreatment of a tumor.
 84. The method according to claim 81, wherein theadenovirus comprises a gene for the treatment of restenosis.
 85. Amethod for the treatment of a disease by gene therapy comprising thestep of administering an adenovirus of claim
 1. 86. (canceled) 87.(canceled)
 88. A pharmaceutical composition comprising an adenovirus ofclaim 1 and an efficient quantity of a pharmaceutically activeexcipient.